Capsular gram-positive bacteria bioconjugate vaccines

ABSTRACT

The present invention encompasses a novel  S. aureus  bioconjugate vaccine. More generally, the invention is directed to Gram-positive and other bioconjugate vaccines containing a protein carrier, at least one polysaccharide such as a capsular Gram-positive polysaccharide, and, optionally, an adjuvant or pharmaceutically acceptable carrier. The instant invention also includes methods of producing Gram-positive and other bioconjugate vaccines. An N-glycosylated protein is also provided that contains one or more polysaccharides such as Gram-positive polysaccharides. The invention is additionally directed to engineered prokaryotic organisms comprising nucleotide sequences encoding a glycosyltransferase of a first prokaryotic organism and a glycosyltransferase of a second prokaryotic organism. The invention further includes plasmids and prokaryotic cells transformed with plasmids encoding polysaccharides and enzymes which produce an N-glycosylated protein and/or bioconjugate vaccine. Further, the invention is directed to methods of inducing an immune response in a mammal comprising administering said bioconjugate vaccines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 61/332,170, filed May 6, 2010, hereinincorporated by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Aspects of this invention were made with government support under grant1R01AI088754-2, subgrant 105699, awarded by the National Institutes ofHealth. The government has certain rights in these aspects of theinvention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is herein incorporated byreference in its entirety. Said ASCII copy, created on May 2, 2011, isnamed 031229US.txt and is 206,590 bytes in size.

BACKGROUND OF THE INVENTION

Vaccines have been one of the great public health inventions of modernmedicine and have saved millions of lives. Immunizations have beenproven to be an ideal means to prevent and control infections. Each yearvaccines prevent up to 3 million deaths and 750,000 children are savedfrom disability. (Global Alliance for Vaccines and Immunization—PressReleases (Mar. 11, 2006) atwww.gavialliance.org/media_centre/press_releases/2006_03_09_enpr_queenrania_delhi.php).In 1999 the CDC declared immunizations the number one public healthachievement of the 20^(th) century (Ten great public healthachievements-United States, 1900-1999. MMWR Morb Mortal Wkly Rep48:241-3 (Apr. 2, 1999)). Some bacteria like those causing tetanus ordiphtheria produce a toxin that is largely responsible for the disease.This toxin can be used in a detoxified form as vaccine. However, formost bacteria there is no single toxin that can be used to develop avaccine.

Among the most successful vaccines are surface polysaccharides ofbacterial pathogens like Haemophilus influenzae, Neisseria meningitidis,and Streptococcus pneumoniae conjugated to carrier proteins. Thesebacteria are surrounded by a capsule, which promotes microbial virulenceand resistance to phagocytic killing, as well as preventing them fromdesiccation.

Bacterial polysaccharides can elicit a long-lasting immune response inhumans if they are coupled to a protein carrier that contains T-cellepitopes. This concept was elaborated 80 years ago (Avery, O. T., and W.F. Goebel. 1929. Chemo-immunological studies on conjugatedcarbohydrate-proteins. II Immunological specificity of syntheticsugar-proteins. J. Exp. Med. 50:521-533), and proven later for thepolysaccharide of Haemophilus influenza type B (HIB) coupled to theprotein carrier diphtheria toxin (Anderson, P. 1983. Antibody responsesto Haemophilus influenzae type b and diphtheria toxin induced byconjugates of oligosaccharides of the type b capsule with the nontoxicprotein CRM197. Infect Immun 39:233-8; Schneerson, R., O. Barrera, A.Sutton, and J. B. Robbins. 1980. Preparation, characterization, andimmunogenicity of Haemophilus influenzae type b polysaccharide-proteinconjugates. J Exp Med 152:361-76). This glycoconjugate was also thefirst conjugated vaccine to be licensed in the USA in 1987 andintroduced into the US infant immunization schedule shortly thereafter.Besides HIB, conjugated vaccines were successfully used against theencapsulated human pathogens N. meningitidis and S. pneumoniae. Routineuse of these vaccines has resulted in decreased nasopharyngealcolonization, as well as infection. Currently approximately ˜25% of theglobal vaccine market comprises conjugated vaccines.

Gram-positive bacteria have a cell membrane that is surrounded bycapsular polysaccharides. Staphylococcus is one such Gram-positivebacterium.

Staphylococcus aureus causes infection. S. aureus is an opportunisticbacterial pathogen responsible for a diverse spectrum of human diseases.Although S. aureus may colonize mucosal surfaces of normal humans, it isalso a major cause of wound infections and has the invasive potential toinduce severe infections, including osteomyelitis, endocarditis, andbacteremia with metastatic complications (Lowy, F. D. 1998.Staphylococcus aureus infections. New Engl J Med 339:520-32). S. aureusis one of the most common agents implicated in ventilator-associatedpneumonia, and it is an important and emerging cause ofcommunity-acquired pneumonia, affecting previously healthy adults andchildren lacking predisposing risk factors (Kollef, M. H., A. Shorr, Y.P. Tabak, V. Gupta, L. Z. Liu, and R. S. Johannes. 2005. Epidemiologyand outcomes of health-care-associated pneumonia: results from a largeUS database of culture-positive pneumonia. Chest 128:3854-62; Shorr, A.F. 2007. Epidemiology and economic impact of meticillin-resistantStaphylococcus aureus: review and analysis of the literature.Pharmacoeconomics 25:751-68).

S. aureus is the second most common cause of nosocomial bacteremia, andmethicillin-resistant S. aureus (MRSA) strains account for more than 50%of all infections in intensive care units in the U.S. S. aureusinfections within the hospital and in the community are increasing. MRSAstrains were isolated from 2% of staphylococcal infections in 1974 andfrom 63% of staphylococcal infections in 2004. Many of the nosocomialMRSA strains are multi-drug resistant, and even methicillin-sensitivestrains can be deadly. A recent report using population-based, activecase finding revealed that 94,360 invasive MRSA infections occurred inthe U.S. in 2005, and that the majority of these (58%) occurred outsideof the hospital (Klevens, R. M., M. A. Morrison, J. Nadle, S. Petit, K.Gershman, S. Ray, L. H. Harrison, R. Lynfield, G. Dumyati, J. M. Townes,A. S. Craig, E. R. Zell, G. E. Fosheim, L. K. McDougal, R. B. Carey, andS. K. Fridkin. 2007. Invasive methicillin-resistant Staphylococcusaureus infections in the United States. JAMA 298:1763-71). In thisanalysis, more Americans died from MRSA (>18,000 deaths) in 2005 thanfrom AIDS.

S. aureus USA100, also known as the New York/Japan clone, is an MRSAstrain that represents the predominant U.S. hospital-acquired MRSAstrain (McDougal, L. K., C. D. Steward, G. E. Killgore, J. M. Chaitram,S. K. McAllister, and F. C. Tenover. 2003. Pulsed-field gelelectrophoresis typing of oxacillin-resistant Staphylococcus aureusisolates from the United States: establishing a national database. JClin Microbiol 41:5113-20).

Epidemiologic analyses indicate that S. aureus causes approximately 2million clinical infections each year in the U.S. alone (Fridkin, S. K.,J. C. Hageman, M. Morrison, L. T. Sanza, K. Como-Sabetti, J. A.Jernigan, K. Harriman, L. H. Harrison, R. Lynfield, and M. M. Farley.2005. Methicillin-resistant Staphylococcus aureus disease in threecommunities. N Engl J Med 352:1436-44; King, M. D., B. J. Humphrey, Y.F. Wang, E. V. Kourbatova, S. M. Ray, and H. M. Blumberg. 2006.Emergence of community-acquired methicillin-resistant Staphylococcusaureus USA 300 clone as the predominant cause of skin and soft-tissueinfections. Ann Intern Med 144:309-17; Klevens, R. M., M. A. Morrison,J. Nadle, S. Petit, K. Gershman, S. Ray, L. H. Harrison, R. Lynfield, G.Dumyati, J. M. Townes, A. S. Craig, E. R. Zell, G. E. Fosheim, L. K.McDougal, R. B. Carey, S. K. Fridkin, and M. I. for the Active BacterialCore surveillance. 2007. Invasive methicillin-resistant Staphylococcusaureus infections in the United States. JAMA 298:1763-1771). Not onlyare S. aureus infections increasing in number, but the resistance of S.aureus to antibiotics is also on the increase. MRSA accounts for 40%-60%of nosocomial S. aureus infections in the U.S., and many of thesestrains are multi-drug resistant. Notorious as a major source ofnosocomial infections, S. aureus has recently taken on a new role incausing an escalating number of community-acquired infections innon-hospitalized persons without predisposing risk factors. Virulentcommunity-associated MRSA (CA-MRSA) strains are becoming more prevalentacross the U.S. and Europe, and their dissemination has been observedglobally (Baggett, H. C., T. W. Hennessy, K. Rudolph, D. Bruden, A.Reasonover, A. Parkinson, R. Sparks, R. M. Donlan, P. Martinez, K.Mongkolrattanothai, and J. C. Butler. 2004. Community-onsetmethicillin-resistant Staphylococcus aureus associated with antibioticuse and the cytotoxin Panton-Valentine leukocidin during a furunculosisoutbreak in rural Alaska. J Infect Dis 189:1565-73; Gilbert, M., J.MacDonald, D. Gregson, J. Siushansian, K. Zhang, S. Elsayed, K.Laupland, T. Louie, K. Hope, M. Mulvey, J. Gillespie, D. Nielsen, V.Wheeler, M. Louie, A. Honish, G. Keays, and J. Conly. 2006. Outbreak inAlberta of community-acquired (USA300) methicillin-resistantStaphylococcus aureus in people with a history of drug use, homelessnessor incarceration. Canad Med Assoc J 175:149-54; Kazakova, S. V., J. C.Hageman, M. Matava, A. Srinivasan, L. Phelan, B. Garfinkel, T. Boo, S.McAllister, J. Anderson, B. Jensen, D. Dodson, D. Lonsway, L. K.McDougal, M. Arduino, V. J. Fraser, G. Killgore, F. C. Tenover, S. Cody,and D. B. Jernigan. 2005. A clone of methicillin-resistantStaphylococcus aureus among professional football players. N Engl J Med352:468-75).

Not only has S. aureus resistance to methicillin become more common, butnumerous isolates with reduced susceptibility to vancomycin have beenreported. Seven clinical isolates of S. aureus that carry vanA and arefully resistant to vancomycin have been isolated in the U.S. Theseisolates are also methicillin resistant (Chang, S., D. M. Sievert, J. C.Hageman, M. L. Boulton, F. C. Tenover, F. P. Downes, S. Shah, J. T.Rudrik, G. R. Pupp, W. J. Brown, D. Cardo, and S. K. Fridkin. 2003.Infection with vancomycin-resistant Staphylococcus aureus containing thevanA resistance gene. New Engl J Med 348:1342-7). Because S. aureuscannot always be controlled by antibiotics and MRSA isolates arebecoming increasingly prevalent in the community, additional controlstrategies, such as a vaccine, are sorely needed.

S. aureus capsular polysaccharides are involved in infection. Manyvirulence factors contribute to the pathogenesis of staphylococcalinfections, including surface-associated adhesions, secreted exoproteinsand toxins, and immune evasion factors (Foster, T. J. 2005. Immuneevasion by staphylococci. Nature Reviews Microbiology 3:948-58). Likemany invasive bacterial pathogens, S. aureus produces a capsularpolysaccharide (CP) (FIG. 4) that enhances its resistance to clearanceby host innate immune defenses. Most clinical isolates of S. aureus areencapsulated, and serotype 5 and 8 strains predominate (Arbeit, R. D.,W. W. Karakawa, W. F. Vann, and J. B. Robbins. 1984. Predominance of twonewly described capsular polysaccharide types among clinical isolates ofStaphylococcus aureus. Diagn Microbiol Infect Dis 2:85-91). The type 5(CP5) and type 8 (CP8) capsular polysaccharides have similartrisaccharide repeating units comprised of N-acetyl mannosaminuronicacid (ManNAcA), N-acetyl L-fucosamine (L-FucNAc), and N-acetylD-fucosamine (D-FucNAc) (Jones, C. 2005. Revised structures for thecapsular polysaccharides from Staphylococcus aureus types 5 and 8,components of novel glycoconjugate vaccines. Carbohydr Res340:1097-106). CP5 and CP8 are serologically distinct, and this can beattributed to differences in the linkages between the sugars and in thesites of O-acetylation (FIG. 4).

Previous studies have correlated S. aureus capsule production withresistance to in vitro phagocytic uptake and killing (Fattom, A., R.Schneerson, S. C. Szu, W. F. Vann, J. Shiloach, W. W. Karakawa, and J.B. Robbins. 1990. Synthesis and immunologic properties in mice ofvaccines composed of Staphylococcus aureus type 5 and type 8 capsularpolysaccharides conjugated to Pseudomonas aeruginosa exotoxin A. InfectImmun 58:2367-74; Thakker, M., J.-S. Park, V. Carey, and J. C. Lee.1998. Staphylococcus aureus serotype 5 capsular polysaccharide isantiphagocytic and enhances bacterial virulence in a murine bacteremiamodel. Infect Immun 66:5183-5189; Watts, A., D. Ke, Q. Wang, A. Pillay,A. Nicholson-Weller, and J. C. Lee. 2005. Staphylococcus aureus strainsthat express serotype 5 or serotype 8 capsular polysaccharides differ invirulence. Infect Immun 73:3502-11). Human neutrophils phagocytosecapsule-negative mutants in the presence of nonimmune serum withcomplement activity, whereas encapsulated isolates require bothcapsule-specific antibodies and complement for optimal opsonophagocytickilling (Bhasin, N., A. Albus, F. Michon, P. J. Livolsi, J.-S. Park, andJ. C. Lee. 1998. Identification of a gene essential for O-acetylation ofthe Staphylococcus aureus type 5 capsular polysaccharide. Mol Microbiol27:9-21; Thakker, M., J.-S. Park, V. Carey, and J. C. Lee. 1998.Staphylococcus aureus serotype 5 capsular polysaccharide isantiphagocytic and enhances bacterial virulence in a murine bacteremiamodel. Infect Immun 66:5183-5189; Watts, A., D. Ke, Q. Wang, A. Pillay,A. Nicholson-Weller, and J. C. Lee. 2005. Staphylococcus aureus strainsthat express serotype 5 or serotype 8 capsular polysaccharides differ invirulence. Infect Immun 73:3502-11). Nilsson et al. (Nilsson, I.-M., J.C. Lee, T. Bremell, C. Ryden, and A. Tarkowski. 1997. The role ofstaphylococcal polysaccharide microcapsule expression in septicemia andseptic arthritis. Infect Immun 65:4216-4221) reported that peritonealmacrophages from mice phagocytosed significantly greater numbers of aCP5-negative mutant compared to the parental strain Reynolds. Oncephagocytosed, the CP5-positive strain survived intracellularly to agreater extent than the mutant strain. Cunnion et al. (Cunnion, K. M.,J. C. Lee, and M. M. Frank. 2001. Capsule production and growth phaseinfluence binding of complement to Staphylococcus aureus. Infect Immun69:6796-6803) compared opsonization of isogenic S. aureus strains anddemonstrated that the CP5-positive strain bound 42% less serumcomplement (C′) than the acapsular mutant.

S. aureus vaccine development conventionally has involved the capsule asa target. Vaccine design for protection against staphylococcal diseaseis complicated by the protean manifestations and clinical complexity ofS. aureus infections in humans. Many S. aureus vaccine candidates havebeen investigated in animal models of infection, but it has beenreported that only two immunization regimens have completed phase IIIclinical trials (Schaffer, A. C., and J. C. Lee. 2008. Vaccination andpassive immunisation against Staphylococcus aureus. Int J AntimicrobAgents 32 Suppl 1:S71-8). The first vaccine is based on the two capsularpolysaccharides (CPs) (FIG. 4) that are most prevalent among clinicalstrains of S. aureus. Fattom et al. (Fattom, A. R. Schneerson, S. C.Szu, W. F. Vann, J. Shiloach, W. W. Karakawa and J. B. Robbins. 1990.Synthesis and immunologic properties in mice of vaccines composed ofStaphylococcus aureus type 5 and type 8 capsular polysaccharidesconjugated to Pseudomonas aeruginosa exotoxin. Infect Immun 58: 2367-74)conjugated the serotype 5 (CP5) and serotype 8 (CP8) polysaccharides tonontoxic recombinant P. aeruginosa exoprotein A (rEPA). The conjugatevaccines were immunogenic in mice and humans, and they induced opsonicantibodies that showed efficacy in protecting rodents from lethality andfrom nonlethal staphylococcal infection (Fattom, A. R. Schneerson, S. C.Szu, W. F. Vann, J. Shiloach, W. W. Karakawa and J. B. Robbins. 1990.Synthesis and immunologic properties in mice of vaccines composed ofStaphylococcus aureus type 5 and type 8 capsular polysaccharidesconjugated to Pseudomonas aeruginosa exotoxin. Infect Immun 58: 2367-74;Fattom, A., R. Schneerson, D. C. Watson, W. W. Karakawa, D. Fitzgerald,I. Pastan, X. Li, J. Shiloach, D. A. Bryla, and J. B. Robbins. 1993.Laboratory and clinical evaluation of conjugate vaccines composed of S.aureus type 5 and type 8 capsular polysaccharides bound to Pseudomonasaeruginosa recombinant exoprotein A. Infect Immun 61:1023-32; Fattom, A.I., J. Sarwar, A. Ortiz, and R. Naso. 1996. A Staphylococcus aureuscapsular polysaccharide (CP) vaccine and CP-specific antibodies protectmice against bacterial challenge. Infect Immun 64:1659-65; Lee, J. C.,J. S. Park, S. E. Shepherd, V. Carey, and A. Fattom. 1997. Protectiveefficacy of antibodies to the Staphylococcus aureus type 5 capsularpolysaccharide in a modified model of endocarditis in rats. Infect Immun65:4146-51). Passive immunization studies have indicated that both CP5-and CP8-specific antibodies significantly reduce infection in a murinemodel of S. aureus mastitis (Tuchscherr, L. P., F. R. Buzzola, L. P.Alvarez, J. C. Lee, and D. O. Sordelli. 2008. Antibodies to capsularpolysaccharide and clumping factor A prevent mastitis and the emergenceof unencapsulated and small-colony variants of Staphylococcus aureus inmice. Infect Immun 76:5738-44). The combined CP5- and CP8-conjugatevaccine was shown to be safe in humans and elicited antibodies thatshowed opsonophagocytic activity.

S. aureus vaccine development has also involved surface proteins as atarget. The second S. aureus clinical vaccine trial was based on theprotective efficacy of antibodies to staphylococcal adhesions inpreventing staphylococcal infections. S. aureus clumping factor A is acell wall-anchored protein that is surface expressed, mediatesstaphylococcal adherence to fibrinogen (Foster, T. J., and M. Hook.1998. Surface protein adhesins of Staphylococcus aureus. TrendsMicrobiol 6:484-8), and promotes the attachment of S. aureus tobiomaterial surfaces (Vaudaux, P. E., P. Francois, R. A. Proctor, D.McDevitt, T. J. Foster, R. M. Albrecht, D. P. Lew, H. Wabers, and S. L.Cooper. 1995. Use of adhesion-defective mutants of Staphylococcus aureusto define the role of specific plasma proteins in promoting bacterialadhesion to canine arteriovenous shunts. Infection & Immunity63:585-90), blood clots, and damaged endothelial surfaces (Moreillon,P., J. M. Entenza, P. Francioli, D. McDevitt, T. J. Foster, P. Francois,and P. Vaudaux. 1995. Role of Staphylococcus aureus coagulase andclumping factor in pathogenesis of experimental endocarditis. Infection& Immunity 63:4738-43). The fibrinogen-binding domain of ClfA is locatedwithin region A of the full-length protein (McDevitt, D., P. Francois,P. Vaudaux, and T. J. Foster. 1995. Identification of the ligand-bindingdomain of the surface-located fibrinogen receptor (clumping factor) ofStaphylococcus aureus. Molecular Microbiology 16:895-907). ClfA plays animportant role in S. aureus binding to platelets, an interaction that iscritical in animal models of catheter-induced staphylococcalendocarditis (Sullam, P. M., A. S. Bayer, W. M. Foss, and A. L. Cheung.1996. Diminished platelet binding in vitro by Staphylococcus aureus isassociated with reduced virulence in a rabbit model of infectiveendocarditis. Infection & Immunity 64:4915-21).

Nanra et al. reported that antibodies to ClfA induced opsonophagocytickilling of S. aureus in vitro (Nanra, J. S., Y. Timofeyeva, S. M.Buitrago, B. R. Sellman, D. A. Dilts, P. Fink, L. Nunez, M. Hagen, Y. V.Matsuka, T. Mininni, D. Zhu, V. Pavliak, B. A. Green, K. U. Jansen, andA. S. Anderson. 2009. Heterogeneous in vivo expression of clumpingfactor A and capsular polysaccharide by Staphylococcus aureus:Implications for vaccine design. Vaccine 27:3276-80). Furthermore, miceimmunized with a recombinant form of the binding region A of ClfA showedreductions in arthritis and lethality induced by S. aureus (Josefsson,E., O. Hartford, L. O'Brien, J. M. Patti, and T. Foster. 2001.Protection against experimental Staphylococcus aureus arthritis byvaccination with clumping factor A, a novel virulence determinant.Journal of Infectious Diseases 184:1572-80). Passive immunizationexperiments were performed in rabbits given a human polyclonalimmunoglobulin preparation that contained elevated levels of antibodiesspecific for ClfA (Vernachio, J., A. S. Bayer, T. Le, Y. L. Chai, B.Prater, A. Schneider, B. Ames, P. Syribeys, J. Robbins, J. M. Patti, J.Vernachio, A. S. Bayer, T. Le, Y.-L. Chai, B. Prater, A. Schneider, B.Ames, P. Syribeys, J. Robbins, and J. M. Patti. 2003. Anti-clumpingfactor A immunoglobulin reduces the duration of methicillin-resistantStaphylococcus aureus bacteremia in an experimental model of infectiveendocarditis. Antimicrobial Agents & Chemotherapy 47:3400-6). Thecombination therapy resulted in better bacterial clearance from theblood of rabbits with catheter-induced S. aureus endocarditis than didvancomycin treatment alone. In addition, passive transfer ofClfA-specific antibodies significantly reduced infection in a murinemodel of S. aureus mastitis (Tuchscherr, L. P., F. R. Buzzola, L. P.Alvarez, J. C. Lee, and D. O. Sordelli. 2008. Antibodies to capsularpolysaccharide and clumping factor A prevent mastitis and the emergenceof unencapsulated and small-colony variants of Staphylococcus aureus inmice. Infect Immun 76: 5738-44).

A phase III clinical trial was reportedly designed to protect againstlate-onset sepsis in 2000 low birth weight, premature neonates. Theinfants received up to four administrations of Veronate, a humanimmunoglobulin preparation pooled from donors with elevated antibodytiters against ClfA and SdrG. Despite the promising results from asimilar phase II clinical trial, this prophylactic therapy resulted inno reduction in the frequency of staphylococcal infections in theneonates (DeJonge, M., D. Burchfield, B. Bloom, M. Duenas, W. Walker, M.Polak, E. Jung, D. Millard, R. Schelonka, F. Eyal, A. Morris, B. Kapik,D. Roberson, K. Kesler, J. Patti, and S. Hetherington. 2007. Clinicaltrial of safety and efficacy of INH-A21 for the prevention of nosocomialstaphylococcal bloodstream infection in premature infants. J Pediatr151:260-5).

It has been shown that protein glycosylation occurs, but rarely does sonaturally, in prokaryotic organisms. On the other hand, N-linked proteinglycosylation is an essential and conserved process occurring in theendoplasmic reticulum of eukaryotic organisms. It is important forprotein folding, oligomerization, stability, quality control, sortingand transport of secretory and membrane proteins (Helenius, A., andAebi, M. (2004). Roles of N-linked glycans in the endoplasmic reticulum.Annu. Rev. Biochem. 73, 1019-1049). Protein glycosylation has aprofoundly favorable influence on the antigenicity, the stability andthe half-life of a protein. In addition, glycosylation can assist thepurification of proteins by chromatography, e.g. affinity chromatographywith lectin ligands bound to a solid phase interacting with glycosylatedmoieties of the protein. It is therefore established practice to producemany glycosylated proteins recombinantly in eukaryotic cells to providebiologically and pharmaceutically useful glycosylation patterns.

Conjugate vaccines have been successfully used to protect againstbacterial infections. The conjugation of an antigenic polysaccharide toa protein carrier is required for protective memory response, aspolysaccharides are T-cell independent antigens. Polysaccharides havebeen conjugated to protein carriers by different chemical methods, usingactivation reactive groups in the polysaccharide as well as the proteincarrier. (Qian, F., Y. Wu, O. Muratova, H. Zhou, G. Dobrescu, P. Duggan,L. Lynn, G. Song, Y. Zhang, K. Reiter, N. MacDonald, D. L. Narum, C. A.Long, L. H. Miller, A. Saul, and G. E. Mullen. 2007. Conjugatingrecombinant proteins to Pseudomonas aeruginosa ExoProtein A: a strategyfor enhancing immunogenicity of malaria vaccine candidates. Vaccine25:3923-3933; Pawlowski, A., G. Kallenius, and S. B. Svenson. 2000.Preparation of pneumococcal capsular polysaccharide-protein conjugatesvaccines utilizing new fragmentation and conjugation technologies.Vaccine 18:1873-1885; Robbins, J. B., J. Kubler-Kielb, E. Vinogradov, C.Mocca, V. Pozsgay, J. Shiloach, and R. Schneerson. 2009. Synthesis,characterization, and immunogenicity in mice of Shigella sonneiO-specific oligosaccharide-core-protein conjugates. Proc Natl Acad SciUSA 106:7974-7978).

Conjugate vaccines can be administered to children to protect themagainst bacterial infections and can provide a long lasting immuneresponse to adults. Constructs of the invention have been found togenerate an IgG response in animals. It is believed that thepolysaccharide (i.e. sugar residue) triggers a short-term immuneresponse that is sugar-specific. Indeed, the human immune systemgenerates a strong response to specific polysaccharide surfacestructures of bacteria, such as O-antigens and capsular polysaccharides.However, as the immune response to polysaccharides is IgM dependent, theimmune system develops no memory. The protein carrier that carries thepolysaccharide, however, triggers an IgG response that is T-celldependent and that provides long lasting protection since the immunesystem develops memory. For this reason, in developing a vaccine, it isadvantageous to develop it as a protein carrier-polysaccharideconjugate.

Prokaryotic organisms rarely produce glycosylated proteins. However, ithas been demonstrated that a bacterium, the food-borne pathogenCampylobacter jejuni, can glycosylate its proteins (Szymanski, et al.(1999). Evidence for a system of general protein glycosylation inCampylobacter jejuni. Mol. Microbiol. 32, 1022-1030). The machineryrequired for glycosylation is encoded by 12 genes that are clustered inthe pgl locus. Disruption of glycosylation affects invasion andpathogenesis of C. jejuni but is not lethal as in most eukaryoticorganisms (Burda P. and M. Aebi, (1999). The dolichol pathway ofN-linked glycosylation. Biochim Biophys Acta 1426(2):239-57). It hasbeen shown that the pgl locus is responsible for N-linked proteinglycosylation in Campylobacter and that it is possible to reconstitutethe N-glycosylation of C. jejuni proteins by recombinantly expressingthe pgl locus and acceptor glycoprotein in E. coli at the same time(Wacker, M., D. Linton, P. G. Hitchen, M. Nita-Lazar, S. M. Haslam, S.J. North, M. Panico, H. R. Morris, A. Dell, B. W. Wren, and M. Aebi.2002. N-linked glycosylation in C. jejuni and its functional transferinto E. coli. Science 298:1790-3).

The N-linked protein glycosylation biosynthetic pathway of Campylobacterhas significant similarities to the polysaccharide biosynthesis pathwayin bacteria (Bugg, T. D., and P. E. Brandish. 1994. From peptidoglycanto glycoproteins: common features of lipid-linked oligosaccharidebiosynthesis. FEMS Microbiol Lett 119:255-62). Based on the knowledgethat antigenic polysaccharides of bacteria and the oligosaccharides ofCampylobacter are both synthesized on the carrier lipid, undecaprenylpyrophosphate (UndPP), the two pathways were combined in E. coli(Feldman, M. F., M. Wacker, M. Hernandez, P. G. Hitchen, C. L. Marolda,M. Kowarik, H. R. Morris, A. Dell, M. A. Valvano, and M. Aebi. 2005.Engineering N-linked protein glycosylation with diverse O antigenlipopolysaccharide structures in Escherichia coli. Proc Natl Acad SciUSA 102:3016-21). It was demonstrated that PglB does not have a strictspecificity for the lipid-linked sugar substrate. The antigenicpolysaccharides assembled on UndPP are captured by PglB in the periplasmand transferred to a protein carrier (Feldman, M. F., M. Wacker, M.Hernandez, P. G. Hitchen, C. L. Marolda, M. Kowarik, H. R. Morris, A.Dell, M. A. Valvano, and M. Aebi. 2005. Engineering N-linked proteinglycosylation with diverse O antigen lipopolysaccharide structures inEscherichia coli. Proc Natl Acad Sci USA 102:3016-21; Wacker, M., M. F.Feldman, N. Callewaert, M. Kowarik, B. R. Clarke, N. L. Pohl, M.Hernandez, E. D. Vines, M. A. Valvano, C. Whitfield, and M. Aebi. 2006.Substrate specificity of bacterial oligosaccharyltransferase (OTase)suggests a common transfer mechanism for the bacterial and eukaryoticsystems. Proc Natl Acad Sci USA 103:7088-93). It was shown thatCampylobacter PglB transfers a diverse array of UndPP linkedoligosaccharides if they contain an N-acetylated hexosamine at thereducing terminus (Wacker et al. (2006)), allowing conjugation of anantigenic polysaccharide to a protein of choice through an N-glycosidiclinkage. While this may provide a theoretical basis for production ofconjugated vaccines in vivo, many difficult challenges need to beovercome in order to realize this theoretical possibility.

Based on this previous discovery that C. jejuni contains a generalN-linked protein glycosylation system, E. coli had been modified toinclude the N-linked protein glycosylation machinery of C. jejuni. Inthis way, glycosylated forms of proteins native to C. jejuni in an E.coli host were produced. It had been further shown that this processcould be used to produce glycosylated proteins from different origins inmodified E. coli host for use as vaccine products. Production by E. coliis advantageous because large cultures of such modified E. coli hostscan be produced which produce large quantities of useful vaccine.

Using this process to produce a glycosylated protein in a modified E.coli host for use as a vaccine product for S. aureus encounters problemsthat have been perceived to be insurmountable. First, E. coli is aGram-negative bacterium and its saccharide biosynthesis pathways differgreatly from those of a Gram-positive bacterium, such as S. aureus,after the polymerization step. In addition, it would have beeninfeasible to genetically engineer E. coli to produce the S. aureuscapsular polysaccharide directly consistent with previous technologies.For example, S. aureus is a Gram positive organism and its capsulesynthesis is associated with cell envelope structure and construction ofthe cellular hull. The capsule producing biosynthetic machinery isspecifically designed to arrange the capsular polysaccharide (PS) on theoutside of the cell and its cell wall. It would have been extremelydifficult, for at least the reason that it would be highlyresource-intensive, to produce this capsule in a modified E. coliorganism, because the cell envelope of E. coli is constructed in afundamentally different way. The biosynthetic machinery for capsuleassembly from PS precursor would be non-functional due to the differentenvironment. Whereas the S. aureus capsule must transit a singlemembrane, in E. coli there is an additional membrane which needs to becrossed to reach the final location of an authentic capsule.Furthermore, as the S. aureus capsule is very large, it was believed tobe infeasible to make a large capsule like the S. aureus capsule betweenthe two membranes of E. coli.

The principle that enzymes from different organisms can work togetherhas been shown before (e.g. Rubires, X., F. Saigi, N. Pique, N. Climent,S. Merino, S. Alberti, J. M. Tomas and M. Regue. 1997. A gene (wbbL)from Serratia marcescens N28b (04) complements the rfb-50 mutation ofEscherichia coli K-12 derivatives. J. Bacteriol 179(23): 7581-6).However, it is believed that no modified LPS polysaccharide from aGram-positive organism has previously been produced in a Gram-negativeorganism.

BRIEF SUMMARY OF THE INVENTION

We have now surprisingly discovered a novel S. aureus bioconjugatevaccine. This novel S. aureus vaccine is based on the novel andunexpected discovery that an oligo- or polysaccharide of a prokaryotehaving one Gram strain can glycosylate a protein in a host prokaroytehaving a different Gram strain. Further novel and unexpected features ofthe invention include without limitation the embodiments set forthbelow.

More generally, the present invention is directed to a bioconjugatevaccine, such as a Gram-positive vaccine, comprising a protein carriercomprising an inserted nucleic acid consensus sequence; at least oneoligo- or polysaccharide from a bacterium such as a Gram-positivebacterium linked to the consensus sequence, and, optionally, anadjuvant. Further, the invention is directed to a Gram-positive bacteriavaccine, such as an S. aureus vaccine, or other bacteria vaccine, madeby a glycosylation system using a modified LPS biosynthetic pathway,which comprises the production of a modified capsular polysaccharide orLPS.

The instant invention is additionally directed to a recombinantN-glycosylated protein comprising a protein comprising at least oneinserted consensus sequence D/E-X-N-Z-S/T, wherein X and Z may be anynatural amino acid except proline; and at least one oligo- orpolysaccharide from a bacterium such as a Gram-positive bacterium linkedto said consensus sequence.

The present is furthermore directed to a combination of a modifiedcapsular polysaccharide of S. aureus with a protein antigen from thesame organism by N-glycosidic linkage.

The invention is further directed to host prokaryotic organismscomprising a nucleotide sequence encoding one or moreglycosyltransferase of a first prokaryotic species, such as aGram-positive species; one or more glycosyltransferases of a differentprokaryotic species, such as a Gram-negative species; a nucleotidesequence encoding a protein; and a nucleotide sequence encoding anOTase. The invention is additionally directed to an engineered hostprokaryotic organism comprising an introduced nucleotide sequenceencoding glycosyltransferases native only to a Gram-positive prokaryoticorganism; a nucleotide sequence encoding a protein; and a nucleotidesequence encoding an OTase.

The invention is furthermore directed to methods of producing abioconjugate vaccine in a host prokaryotic organism comprising nucleicacids encoding one or more glycosyltransferases of a first prokaryoticspecies, such as a Gram-positive species, for example, S. aureus; one ormore glycosyltransferases of a second prokaryotic species, a protein;and an OTase. In addition, the present invention is directed to theproduction of bioconjugate vaccines by producing in Gram-negativebacteria modified capsular polysaccharides, which can be transferred tolipid A core by WaaL and/or linked to a carrier of choice by the OTase.

The invention is further directed to methods of producing glycosylatedproteins in a host prokaryotic organism comprising nucleotide sequenceencoding glycosyltransferases native to a first prokaryotic organism andalso encoding glycosyltransferases native to a second prokaryoticorganism that is different from the first prokaryotic organism. Thepresent invention is additionally directed to the production of proteinsN-glycosylated with capsular polysaccharides of Gram-positive bacteria,which are synthesized by a combination of different glycosyltransferasesfrom different organisms. The invention is furthermore directed to theproduction of glycosylated proteins in a host prokaryotic organismcomprising an introduced nucleotide sequence encodingglycosyltransferases native only to a Gram-positive prokaryoticorganism.

The instant invention is moreover directed to plasmids, such as,plasmids comprising one or more of SEQ. ID NO: 2, SEQ ID NO: 3 and SEQID NO: 4. The invention also includes plasmids comprising one or more ofSEQ. ID NO: 6; SEQ. ID NO: 7; SEQ. ID NO: 8 and SEQ. ID NO: 16. Theinvention also relates to plasmids comprising one or more of SEQ. ID NO:10; SEQ. ID NO: 11; and SEQ. ID NO: 12. Moreover, the invention isdirected to plasmids comprising one or more of SEQ. ID NO: 13; SEQ. IDNO: 15; SEQ. ID NO: 15; SEQ. ID NO: 17; SEQ ID NO: 18; SEQ. ID NO: 19;SEQ. ID NO: 20; SEQ. ID NO: 21 and SEQ. ID NO: 27.

The invention is additionally directed to transformed bacterial cells,such as, for example, bacterial cells transformed with a plasmidcomprising one or more of SEQ. ID NO. 2; SEQ. ID NO: 3; SEQ. ID NO: 4;SEQ. ID NO: 17; SEQ. ID NO: 18; SEQ. ID NO: 19 and SEQ. ID NO: 20; SEQ.ID NO: 21; and SEQ. ID NO: 27. The instant invention is further directedto a bacterial cell transformed with a plasmid comprising one or more ofSEQ. ID NO: 5; SEQ. ID NO: 8; SEQ. ID NO: 9; SEQ. ID NO: 10; SEQ. ID NO:11; SEQ. ID NO: 12; SEQ. ID NO: 13; SEQ. ID NO: 14; SEQ. ID NO: 15 andSEQ. ID NO: 16.

The instant invention is further directed to a method of inducing animmune response against an infection caused by Gram-positive and otherbacteria in a mammal. In one embodiment, the method comprisesadministering to said mammal an effective amount of a pharmaceuticalcomposition comprising: protein comprising at least one insertedconsensus sequence D/E-X-N-Z-S/T, wherein X and Z may be any naturalamino acid except proline; and one or more oligo- or polysaccharides,the one or more oligo- or polysaccharides being the same or different asanother of the one or more oligo- or polysaccharides, from aGram-positive bacterium linked to said consensus sequence.

In another aspect, the invention features a method of identifying atarget polysaccharide for use in glycosylating a protein with saidtarget polysaccharide, in whole or in part. Said glycosylated proteincomprising the target polysaccharide can be used, for example, invaccine compositions. In one embodiment, the method of identifying atarget polysaccharide includes: identifying a Gram-positive bacterium,such as S. aureus, as a target; identifying a first repeating unit of apolysaccharide produced by said Gram-positive bacterium comprising atleast three monomers; identifying a polysaccharide produced by abacterium of a Gram-negative species comprising a second repeating unitcomprising two of the same monomers as said first repeating unit.

The present invention is also directed to a method for modifying abacterium of a first bacterial species such as a Gram-negative species.In one embodiment, the method includes: identifying a first repeatingunit of a polysaccharide of a Gram-positive species, such as S. aureus,comprising three monomers; identifying a polysaccharide produced by abacterium of a second Gram-negative species comprising another repeatingunit comprising two of the same monomers of the first repeating unit;inserting into said bacterium of a first Gram-negative species one ormore nucleotide sequences encoding glycosyltransferases that assemble atrisaccharide comprising: a) said second repeating unit; and b) amonomer of said first repeating unit not present in said secondrepeating unit; inserting a nucleotide sequence encoding a protein; andinserting a nucleotide sequence encoding an OTase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a pathway for the wzx/wzy-dependent O-antigenbiosynthesis, exemplified by the P. aeruginosa O11 O-antigenbiosynthesis. Protein names putatively responsible for the presentedreactions are indicated above or below the arrows, including uridinediphosphate (UDP) and uridine monophosphate (UMP).

FIG. 2 depicts a proposed pathway for the engineered S. aureus capsularpolysaccharide serotype 5 (CP5) biosynthesis in E. coli. The enzymesprovided by the O-antigen cluster of P. aeruginosa O11 are indicated asin FIG. 1. Enzymes from S. aureus CP5 are indicated as Cap5 (compare toFIG. 6). WecB and WecC are E. coli enzymes required for the productionof UDP-ManNAcA. Other depicted proteins and enzymes include uridinediphosphate (UDP), uridine monophosphate (UMP), and coenzyme A (CoA).

FIG. 3 depicts a proposed pathway for the engineered S. aureus capsularpolysaccharide serotype 8 (CP8) biosynthesis. Gene names are indicatedby arrows (compare to FIGS. 1, 2, and 6). UDP, UMP: uridine diphosphate,uridine monophosphate. CoA: coenzyme A.

FIG. 4 depicts the structural overlap of capsular S. aureus and P.aeruginosa O-antigen Repeating Unit (RU) Structures.

FIG. 5A depicts the SDS-PAGE analysis of the elongation of theincomplete O11 O-antigen RU (repeating unit) by S. aureus enzymes.

FIG. 5B depicts the immunodetection of the elongation of the incompleteO11 O-antigen RU by S. aureus enzymes.

FIG. 6 depicts a strategy in an embodiment of the invention for theconstruction of the chimeric O11/CP5 and O11/CP8 gene clusters.

FIG. 7A depicts polymerized CP5 LPS of an embodiment of the inventiondetected in E. coli lipid extracts.

FIG. 7B depicts polymerized CP8 LPS of an embodiment of the inventiondetected in E. coli lipid extracts.

FIG. 8A depicts recombinant CP5 LPS production of an embodiment of theinvention analyzed by SDS-PAGE and stained by silver in dependence ofantibiotic resistance gene on the pLAFR plasmid containing the chimericcluster in W3110 ΔwecA cells.

FIG. 8B depicts recombinant CP5 LPS production of an embodiment of theinvention analyzed by SDS PAGE, stained by silver and immunodetection independence of antibiotic resistance gene on the pLAFR plasmid containingthe chimeric cluster in W3110 ΔwecA cells.

FIG. 9 depicts recombinant CP5 LPS production of an embodiment of theinvention analyzed SDS PAGE and by immunodetection in dependence ofpromoter in front of the chimeric cluster in W3110 ΔwecA cells.

FIG. 10A shows the results of HPLC analysis of an embodiment ofrecombinant RU of CP5 of the present invention produced using thechimeric CP5 cluster (SEQ ID: 2).

FIG. 10B shows the results of HPLC analysis of an embodiment ofrecombinant RU of CP8 of the present invention produced using a chimericCP8 cluster lacking the cap8I polymerase.

FIG. 11A shows the results of MALDI-MS/MS analysis of the specific peakgenerated by expression of an embodiment of the chimeric CP5 cluster ofthe present invention in E. coli eluting at 37 minutes seen in FIG. 10A.

FIG. 11B shows the results of MALDI-MS/MS analysis of the specific peakgenerated by expression of an embodiment of the chimeric CP5 cluster ofthe present invention in E. coli eluting at 40 minutes seen in FIG. 10A.

FIG. 11C shows the results of MALDI-MS/MS analysis of the specific peakgenerated by expression of an embodiment of the chimeric CP8 cluster ofthe present invention in E. coli eluting at 32 minutes seen in FIG. 10B.

FIG. 11D shows the results of MALDI-MS/MS analysis of the specific peakgenerated by expression of an embodiment of the chimeric CP8 cluster ofthe present invention in E. coli eluting at 38 minutes seen in FIG. 10B.

FIG. 11E shows the results of MALDI-MS/MS analysis of the specific peakgenerated by expression of an embodiment of the chimeric CP8 cluster ofthe present invention in E. coli eluting at 45 minutes seen in FIG. 10B.

FIG. 11F shows the results of HPLC analysis of an embodiment of glycanstructure optimization.

FIG. 11G and FIG. 11G-1 present the results of HPLC analysis of the fullCP5 glycan repertoire present on UndPP in E. coli cells in an embodimentof the present invention.

FIG. 11H presents the results of HPLC analysis of deacetylated CP5glycans and RU homogeneity in an embodiment of the invention.

FIG. 11I provides the results of HPLC analysis of the CP8 glycanrepertoire present on UndPP in E. coli cells in an embodiment of thepresent invention.

FIG. 11J shows HPLC results, in an embodiment of the present invention,of deacetylation of CP8 glycans and RU homogeneity.

FIG. 11K presents HPLC results showing reduction in RU polymerizationand increase in LLO induced by co-expression of wzzO7 with the CP8chimeric cluster in an embodiment of the present invention.

FIG. 12 shows the results of SDS-PAGE analysis of Ni²⁺ affinitychromatography purified EPA-CP5 bioconjugate from cells in embodimentsof the present invention without and with the S. aureus flippase genecap5K (SEQ ID NO: 2 and 3).

FIG. 13A presents analysis of CP5-EPA bioconjugate according to anembodiment of the present invention purified by Ni²⁺ affinitychromatography and anionic exchange chromatography.

FIG. 13B depicts M/Z masses found for the glycosylation site intrypsinized peptide DNNNSTPTVISHR N-glycosidically linked to theO-acetylated RU mass (m/z=2088 ([M+H]⁺)) according to an embodiment ofthe present invention. The inset illustrates the RU structure attachedto the peptide.

FIG. 13C depicts M/Z masses found for the glycosylation site intrypsinized peptide DQNR N-glycosidically linked to the O-acetylated RUmass (m/z=1165 ([M+H]⁺)) according to an embodiment of the presentinvention. The inset illustrates the RU structure attached to thepeptide.

FIG. 13D depicts an analysis of Ni²⁺ affinity chromatography and anionicexchange chromatography purified CP8-EPA bioconjugate according to anembodiment of the present invention.

FIG. 13E depicts purified CP5-EPA bioconjugate from cells containingeither 3 (left) or 2 plasmids (right lane) for glycoconjugate productionaccording to an embodiment of the present invention.

FIG. 13F depicts analysis of Ni²⁺ affinity chromatography purifiedCP8-EPA bioconjugate according to an embodiment of the presentinvention.

FIG. 14A presents High Mass MALDI analysis of a purified CP5-EPAbioconjugate of an embodiment of the invention produced using the 3plasmid system from FIG. 13A.

FIG. 14B shows characterization by size exclusion chromatography ofCP5-EPA bioconjugate of an embodiment of the invention produced usingthe 3 plasmid system from FIG. 13A.

FIG. 14C shows the SDS PAGE analysis and immunodetection of purifiedCP5-Hla bioconjugate according to an embodiment of the presentinvention.

FIG. 14D presents the results of purified CP5-AcrA bioconjugateaccording to an embodiment of the present invention.

FIG. 14E presents the results of purified CP5-ClfA bioconjugateaccording to an embodiment of the present invention.

FIG. 15A depicts the specific anti CP5 antibodies raised in mice byCP5-EPA bioconjugate according to an embodiment of the presentinvention.

FIG. 15B depicts the specific anti CP5 antibodies raised in rabbit byCP5-EPA bioconjugate according to an embodiment of the presentinvention.

FIG. 16A illustrates in vitro opsonophagocytic activity (on S. aureusReynolds) of CP5 specific antibodies raised by immunization of rabbitswith CP5-EPA according to an embodiment of the present invention.

FIG. 16B illustrates in vitro opsonophagocytosis activity (on S. aureusUSA 100) of CP5 specific antibodies raised by immunization of rabbitswith CP5-EPA according to an embodiment of the present invention.

FIG. 17A depicts the results of passive immunization using anti CP5-EPAantibodies, according to an embodiment of the present invention, in micechallenged i.p. with ˜3.6.10⁷ CFU of S. aureus strain Reynolds.

FIG. 17B depicts the results of passive immunization using anti CP5-EPAantibodies, according to an embodiment of invention, in mice injectedwith 2 mg CP5-EPA IgG.

FIG. 17C depicts the results of passive immunization using anti CP5-EPAantibodies, according to an embodiment of the invention, in miceinjected with 300 μg CP5-EPA IgG

FIG. 18 depicts the results of an active immunization assay usingdifferent doses of CP5-EPA as vaccine according to an embodiment of thepresent invention and the mouse bacteremia model for challenge.

DETAILED DESCRIPTION OF THE INVENTION

According to an embodiment of the present invention, an LPSpolysaccharide from a Gram-positive organism has now been shown to beproduced in a Gram-negative organism. We believe that this is a novelresult that represents an important and significant departure from theprior art.

Nucleic acids within the scope of the invention are exemplified by thenucleic acids of the invention contained in the Sequence Listing. Anynucleic acid encoding an immunogenic component, or portion thereof,which is capable of expression in a host cell, can be used in thepresent invention. The following sequence descriptions are provided tofacilitate understanding of certain terms used throughout theapplication and are not to be construed as limiting embodiments of theinvention.

SEQ ID NO: 1 depicts pLAFR1 (Gene Bank Accession AY532632.1) containingthe O11 O-antigen sequence from P. aeruginosa PAO103 in the EcoRI site,complementary strand (partially from Gen Bank Accession AF236052).

SEQ ID NO: 2 depicts pLAFR1 containing the CP5 chimeric cluster,corresponding to the pLAFR1-O11 with the cap5HIJ genes replacingwbjA-wzy by homologous recombination. The inserted sequence alsocontains a cat cassette for selection of homologous recombined clones.

SEQ ID NO: 3 depicts pLAFR1 containing the CP5 chimeric cluster with thecap5K flippase gene, corresponding to the pLAFR-O11 with the cap5HIJgenes replacing wbjA-wzy by homologous recombination and the cap5Kcloned between cap5J and the cat cassette.

SEQ ID NO: 4 depicts pLAFR1 containing the CP8 chimeric clusterincluding a flippase gene, corresponding to the pLAFR1-O11 with thecap8KHIJ genes replacing wbjA-wzy. The inserted sequence also contains acat cassette for selection of homologous recombined clones.

SEQ ID NO: 5 depicts an expression plasmid for Hla H35L production. TheORF encoding Hla H35L is cloned into NdeI/SacI in pEC415.

SEQ ID NO. 6 depicts the expression plasmid for Hla-H35L site 202production. The ORF encodes an N-terminal DsbA signal peptide from E.coli, a glycosite around amino acid position 202, and a C-terminalHIS-tag. This construct is cloned into NheI/SalI on pEC415.

SEQ ID NO: 7 depicts the expression plasmid for Hla-H35L site 238production. The ORF encodes an N-terminal DsbA signal peptide from E.coli, a glycosite around amino acid position 238, and a C-terminalHIS-tag. The above mentioned construct is cloned into NheI/SalI onpEC415.

SEQ ID NO: 8 depicts the expression plasmid for Hla-H35L site 272production. The ORF encodes an N-terminal DsbA signal peptide from E.coli, a glycosite around amino acid position 272, and a C-terminalHIS-tag. The above mentioned construct is cloned into NheI/SalI onpEC415.

SEQ ID NO: 9 depicts an expression plasmid for ClfA production. The genewas chemically synthesized and cloned into the NdeI/SacI in pEC415expression vector.

SEQ ID NO: 10 depicts the expression plasmid for ClfA site 290production. The ORF encodes an N-terminal DsbA signal peptide from E.coli, a glycosite around amino acid position 290, and a C-terminalHIS-tag. The above mentioned construct is cloned into NheI/SalI onpEC415.

SEQ ID NO: 11 depicts the expression plasmid for ClfA site 327production. The ORF encodes an N-terminal DsbA signal peptide from E.coli, a glycosite around amino acid position 327, and a C-terminalHIS-tag. The above mentioned construct is cloned into NheI/SalI onpEC415.

SEQ ID NO: 12 depicts the expression plasmid for ClfA site 532production. The ORF encodes an N-terminal DsbA signal peptide from E.coli, a glycosite around amino acid position 532, and a C-terminalHIS-tag. The above mentioned construct is cloned into NheI/SalI onpEC415.

SEQ ID NO: 13 depicts the amino acid sequence of recombinant,genetically detoxified EPA with a signal sequence and two glycosylationsites at positions 260 and 402.

SEQ ID NO: 14 depicts the amino acid sequence of recombinant,genetically detoxified EPA without signal sequence and two glycosylationsites at positions 241 and 383.

SEQ ID NO: 15 depicts the ORF encoding AcrA cloned via NheI/SalI intopEC415.

SEQ ID NO: 16 depicts the expression plasmid for Hla-H35L site 130production. The ORF encodes an N-terminal DsbA signal peptide from E.coli, a glycosite around amino acid position 130, and a C-terminalHIS-tag. The above mentioned construct is cloned NheI/SalI into pEC415.

SEQ ID NO: 17 depicts CP5 producing gene cluster with cap5K flippasefollowed by a pglB expression cassette consisting of the intergene DNAsequence between galF and wbqA of E. coli serotype O121 and the pglBORF. The insert is cloned in the EcoRI site of pLAFR1.

SEQ ID NO: 18 depicts CP8 producing gene cluster with cap8K flippasefollowed by a pglB expression cassette consisting of the intergene DNAsequence between galF and wbqA of E. coli serotype O121 and the pglBORF. The insert is cloned in the EcoRI site of pLAFR1.

SEQ ID NO: 19 depicts CP8 producing gene cluster with cap8K flippasefollowed by a pglB expression cassette consisting of the intergene DNAsequence between galF and wbqA of E. coli serotype O121 and the pglBORF, in addition this sequence has the gene for wzz of the E. coliserovar O7 cloned into SfaAI/BspTI, i.e. between wzx of Pseudomonasaeruginosa O11 and cap8H. The insert is cloned in the EcoRI site ofpLAFR1.

SEQ ID NO: 20 depicts an expression plasmid for EPA and wzz. Thebackbone is pACT3 in which the resistance cassette was replaced(kanamycin for chloranphenicol)

SEQ ID NO: 21 depicts wzz of E. coli serotype O7 cloned in pext21Eco/Sal.

SEQ ID NO: 22 depicts a peptide sequence set forth in the Examples.

SEQ ID NO: 23 depicts a peptide sequence set forth in the Examples.

SEQ ID NO: 24 depicts a protein consensus sequence, D/E-X-N-Z-S/T,wherein X and Z may be any natural amino acid except proline.

SEQ ID NO: 25 depicts a glycosylation site.

SEQ ID NO: 26 depicts a glycosylation site.

SEQ ID NO: 27 depicts an expression plasmid containing the pglB ORFcloned in EcoRI/BamHI sites.

Descriptions of terms and abbreviations appear below as used in thespecification and consistent with the usages known to one of ordinaryskill in the art. The descriptions are provided to facilitateunderstanding of such terms and abbreviations and are not to beconstrued as limiting embodiments of the invention.

AcrA refers to a glycoprotein from C. jejuni.

Active immunization refers to the induction of immunity (antibodies)after exposure to an antigen.

APCs refers to antigen presenting cells.

Amp refers to ampicillin.

Bacteremia refers to the presence of viable bacteria in the circulatingblood.

C′ refers to complement.

CapA is an enzyme proposed to be a chain length determinant in S. aureusCP5.

CapB is an enzyme proposed to be a regulator of polysaccharide chainlength in S. aureus CP5.

CapC is an enzyme proposed to encode a transporter protein in S. aureusCP5.

CapD an enzyme having 4,6 dehydratase activity and converts theprecursor UDPGlcNAc to UDP-2-acetamido-2,6 dideoxy-D-xylo-4-hexulose inS. aureus CP5.

CapE is a 4,6-dehydratase 3,5-epimerase catalyzing the epimerization ofUDP-D-GlcNAc to UDP-2-acetamido-2, 6-dideoxy-D-lyxo-4-hexulose in S.aureus CP5.

CapF is a reductase, catalyzes the reduction form UDP-2-acetamido-2,6-dideoxy-D-lyxo-4-hexulose to UDP-L-6dTalNAc in S. aureus CP5.

CapG is a 2-Epimerase, catalyzes the epimerization form UDP-L-6dTalNActo UDP-LFucNAc in S. aureus CP5.

CapH in S. aureus CP5 is an O-acetyltransferase.

CapH in CP8 is a transferase similar to CapI from S. aureus CP5.

CapI in S. aureus CP5 is a glycosyltransferase which catalyzes thetransfer of UDP-ManNAcA into carrier lipid-D-FucNAc-L-FucNAc producingcarrier lipid-D-FucNAc-L-FucNAc-ManNAcA.

CapI in CP8 is a polymerase which is similar to CapJ in S. aureus CP5.

CapJ in S. aureus CP5 is a polymerase.

CapJ in CP8 is an O-acetyltransferase similar to CapH in S. aureus CP5.

CapK in S. aureus CP5 is a flippase.

CapK in S. aureus CP8 is a flippase similar to the CapK in CP5.

CapL is a transferase which catalyzes the transfer of UDP-L-FucNAc ontoD-FucNAc-carrier lipid producing carrier lipid-D-FucNAc-L-FucNAc in S.aureus CP5.

CapM is a transferase which catalyzes the transfer of UDP-D-FucNAc on tocarrier lipid producing carrier lipid-D-FucNAc in S. aureus CP5.

CapN is a 4-reductase which catalyzes the reduction fromUDP-2-acetamido-2, 6-dideoxy-D-xylo-4-hexulose. to UDP-D-FucNAc in S.aureus CP5.

CapO is a dehydrogenase which catalyzes the conversion of UDP-D-ManNAcinto UDP-ManNAcA in S. aureus CP5.

CapP is a 2-epimerase which catalyzes the epimerization of UDP-D-GlcNActo UDP-D-ManNAc in S. aureus CP5.

CFU refers to Colony formation unit.

ClfA refers to S. aureus clumping factor A, a cell wall-anchoredprotein.

Conjugate vaccine refers to a vaccine created by covalently attaching apolysaccharide antigen to a carrier protein. Conjugate vaccine elicitsantibacterial immune responses and immunological memory. In infants andelderly people a protective immune response against polysaccharideantigens can be induced if these antigens are conjugated with proteinsthat induce a T-cell dependent response.

Consensus sequence refers to a sequence of amino acids, -D/E-X-N-Z-S/T-wherein X and Z may be any natural amino acid except Proline, withinwhich the site of carbohydrate attachment to N-linked glycoproteins isfound.

Capsular polysaccharide, in its naturally occurring form, refers to athick, mucous-like layer of polysaccharide, is water soluble andcommonly acidic. Naturally-occurring capsular polysaccharides consist ofregularly repeating units of one to several monosaccharides/monomers.

CP5 refers to Staphylococcus aureus type 5 capsular polysaccharide orserotype 5 capsular polysaccharide.

CP8 refers to Staphylococcus aureus type 8 capsular polysaccharide orserotype 8 capsular polysaccharide.

D-FucNAc refers to N-acetyl D-fucosamine.

ECA refers to enterobacterial common antigen.

ELISA refers to Enzyme-linked immunosorbent assay, a biochemicaltechnique used mainly in immunology to detect the presence of anantibody or an antigen in a sample.

EPA or EPAr refers to nontoxic recombinant P. aeruginosa exoprotein A.

Glycoconjugate vaccine refers to a vaccine comprising a protein carrierlinked to an antigenic or immunogenic oligosaccharide.

Glycosyltransferase refers to enzymes that act as a catalyst for thetransfer of a monosaccharide unit from an activated nucleotide sugar toa glycosyl acceptor molecule.

Gram-positive strain refers to a bacterial strain that stains purplewith Gram staining (a valuable diagnostic tool). Gram-positive bacteriahave a thick mesh-like cell wall made of peptidoglycan (approximately50-90% of cell wall).

Gram-negative strain refers to a bacterial strain which has a thinnerlayer (approximately 10% of cell wall) which stains pink. Gram-negativebacteria also have an additional outer membrane that contains lipids,and is separated from the cell wall by the periplasmic space.

Hla (alpha toxin) refers to alpha hemolysin, which is a secretedpore-forming toxin and an essential virulence factor antigen of S.aureus.

Hla H35L refers to a mutant form of Hla nontoxic alpha-toxin mutant fromS. aureus.

Histidine tag, or polyhistidine-tag, is an amino acid motif in proteinsthat consists of at least five histidine (His) residues, often at the N-or C-terminus of the protein, and used to purify in a simple and fastmanner by specifically binding to a nickel affinity column.

IV refers to intravenously.

kDa refers to kilo Daltons, is an atomic mass unit.

L-FucNAc refers to N-acetyl L-fucosamine.

LPS refers to lipopolysaccharide. Lipopolysaccharides (LPS), also knownas lipoglycans, are large molecules consisting of a lipid and apolysaccharide joined by a covalent bond; they are found in the outermembrane of Gram-negative bacteria, act as endotoxins and elicit strongimmune responses in animals.

ManNAcA refers to N-acetyl mannosaminuronic acid.

Methicillin-resistant S. aureus strains (MRSA) refers tomethicillin-resistant S. aureus strain associated with longer hospitalstay and more infections in intensive care units which leads to moreantibiotic administration.

N-glycans or N-linked oligosaccharides refers to mono-, oligo- orpolysaccharides of variable compositions that are linked to an ε-amidenitrogen of an asparagine residue in a protein via an N-glycosidiclinkage.

N-linked protein glycosylation refers to a process or pathway tocovalently link “glycans” (mono-, oligo- or polysaccharides) to anitrogen of asparagine (N) side-chain on a target protein.

O-antigens or O-polysaccharides refers to a repetitive glycan polymercontained within an LPS. The O antigen is attached to the coreoligosaccharide, and comprises the outermost domain of the LPS molecule.

Oligosaccharides or Polysaccharides refers to homo- or heteropolymerformed by covalently bound carbohydrates (monosaccharides), and includesbut is not limited to repeating units (monosaccharides, disaccharides,trisaccharides, etc.) linked together by glycosidic bonds.

Opsonophagocytic activity refers to phagocytosis of a pathogen in thepresence of complement and specific antibodies. The in vitroopsonophagocytic activities (OPAs) of serum antibodies are believed torepresent the functional activities of the antibodies in vivo and thusto correlate with protective immunity.

OTase or OST refers to oligosaccharyl transferase, which catalyzes amechanistically unique and selective transfer of an oligo- orpolysaccharide (glycosylation) to the asparagine (N) residue at theconsensus sequence of nascent or folded proteins.

Passive immunization is the transfer of active humoral immunity in theform of already made antibodies, from one individual to another.

Periplasmic space refers to the space between the inner cytoplasmicmembrane and external outer membrane of Gram-negative bacteria.

PMNs refers to polymorphonuclear neutrophils, which are the mostabundant white blood cells in the peripheral blood of humans, and many(though not all) mammals.

Protein carrier refers to a protein that comprises the consensussequence into which the oligo- or polysaccharide is attached.

RU refers to a repeating unit comprising specific polysaccharidessynthesized by assembling individual monosaccharides into an oligo- orpolysaccharide.

Signal sequence refers to a short (e.g., approximately 3-60 amino acidslong) peptide at the N-terminal end of the protein that directs theprotein to different locations.

UDP-D-ManNAc is UDP-N-acetyl-D-mannosamine.

UDP-D-ManNAcA is UDP-N-acetyl-D-mannosaminuronic acid.

UDP-D-QuiNAc is UDP-N-acetyl-D-quinovosamine.

UDP-L-FucNAc is UDP-N-acetyl-L-fucosamine.

UDP-L-6dTalNAc is UDPN-acetyl-L-pneumosamine.

Und refers to undecaprenyl or undecaprenol lipid composed by elevenprenol units.

UndP refers to undecaprenyl phosphate, which is a universal lipidcarrier (derived from Und) of glycan biosynthetic intermediates forcarbohydrate polymers that are exported to the bacterial cell envelope.

UndPP refers to undecaprenyl pyrophosphate, which is a phosphorylatedversion of UndP.

wbjA is a glucosyltransferase in P. aeruginosa O11.

wbjB is a putative epimerase similar to enzymes required to the capsulebiosynthesis of CP5 and CP8 in S. aureus.

wbjC is a putative epimerase in P. aeruginosa O11.

wbjD is a putative epimerase in P. aeruginosa O11.

wbjE is a putative epimerase in P. aeruginosa O11.

wbjF is a glycosyltranseferase in P. aeruginosa O11.

wbpL is a glycosyltransferase that participates in LPS biosynthesis inP. aeruginosa O11.

wbpM is a glycosyltransferse that participates in LPS biosynthesis in P.aeruginosa O11.

Embodiments of the invention are at least partially based on thediscovery that C. jejuni contains a general N-linked proteinglycosylation system, an unusual feature for prokaryotic organisms.Various proteins of C. jejuni have been shown to be modified by aheptasaccharide. This heptasaccharide is assembled on UndPP, the carrierlipid, at the cytoplasmic side of the inner membrane by the stepwiseaddition of nucleotide activated monosaccharides catalyzed by specificglycosyltransferases. The lipid-linked oligosaccharide is then flippedinto (i.e., it diffuses transversely) the periplasmic space by aflippase, e.g., PglK. In the final step of N-linked proteinglycosylation, the OTase (e.g., PglB) catalyzes the transfer of theoligosaccharide from the carrier lipid to Asn residues within theconsensus sequence Asp/Glu-Xaa-Asn-Zaa-Ser/Thr (i.e., D/E-X-N-Z-S/T),where the Xaa and Zaa can be any amino acid except Pro. We hadsuccessfully transferred the glycosylation cluster for theheptasaccharide into E. coli and were able to produce N-linkedglycoproteins of Campylobacter.

A novel and inventive method to modify a Gram-negative host bacterium,such as E. coli, has been developed to produce glycosylated proteins foruse as vaccine products against a Gram-positive bacterium such as S.aureus. The development of this method required overcoming significantand in many respects unexpected problems, and departing substantiallyfrom conventional wisdom and the prior art.

In this novel and inventive method, another Gram-negative bacterium wasidentified that produces a polysaccharide that has structural similarityto the polysaccharide of interest of the target organism, for example S.aureus. For purposes of this invention, structural similarity manifestsitself as repeating units in the polysaccharide of the target (e.g., S.aureus) that are partially identical to repeating units in thepolysaccharide of the identified, other Gram-negative bacterium. Becausethis latter bacterium is Gram-negative, as is the host, for example, E.coli organism, we initially hypothesized (and later verified byexperiment as discussed below) that use of its biosynthesis pathways ina modified E. coli organism would allow the biosynthesis of theconstructed RU antigen and its flipping from the cytoplasm into theperiplasm of the modified E. coli organism. Further, we hypothesized(and later verified by experiment as discussed below) that the size ofthe polysaccharide produced through this biosynthesis pathway would bemuch smaller than the polysaccharide produced by the biosynthesispathway of Gram positive S. aureus.

As a result, and as discussed below, the novel and innovative method wedeveloped solved the aforementioned difficult problems.

Furthermore, it was surprisingly found that aspects of the LPS pathwayin a Gram-negative organism could be used to produce polysaccharidesthat contain some of the same repeating units as capsularpolysaccharides native to Gram-positive bacteria, such as, for example,S. aureus, as detailed below.

Therefore, in making the polysaccharide section of the glycosylatedprotein vaccine for S. aureus, one surprising solution is to constructthe polysaccharide section at least partially based on a polysaccharidenative to a Gram-negative bacterium like E. coli. We further discoveredthat, in doing so, it is apparently important to find a bacterium whichproduces a polysaccharide that is as similar as possible to thepolysaccharide of interest produced by S. aureus. P. aeruginosa is sucha bacterium.

FIG. 1 provides a step-by-step depiction of an embodiment of thepreparation of nucleotide-activated monosaccharides in the cytoplasmeither by enzymes provided in the O-antigen cluster or by house keepingenzymes of the Gram-negative host cell, as would be apparent to one ofskill in the art in light of this specification. The steps of theprocess proceed from left to right in the depiction of FIG. 1. In theembodiment depicted in FIG. 1, a glycosylphosphate transferase (WbpL)adds D-FucNAc phosphate to UndP, forming UndPP-FucNAc. Specificglycosyltransferases then elongate the UndPP-D-FucNAc molecule furtherby adding monosaccharides forming the repeating unit (RU)oligosaccharide (WbjE, WbjA). The RU is then flipped into theperiplasmic space by the Wzx protein. The Wzy enzyme polymerizesperiplasmic RUs to form the O-antigen polysaccharide. Polymer length iscontrolled by the Wzz protein. Many bacterial oligo- and polysaccharidesare assembled on UndPP and then transferred to other molecules. In otherwords, UndPP is a general building platform for sugars in bacteria. InE. coli and, it is believed, most other Gram negative bacteria, theO-antigen is transferred from UndPP to Lipid A core by the E. colienzyme WaaL to form lipopolysaccharide (LPS).

FIG. 2 depicts an embodiment of preparation of nucleotide-activatedmonosaccharides in the cytoplasm by enzymes provided in the O-antigencluster of P. aeruginosa O11, by house keeping enzymes of theGram-negative host cell, and by S. aureus and/or E. coli enzymes knownto be required for UDP-ManNAcA biosynthesis (Cap5OP and/or WecBC), aswould be apparent to one of skill in the art in light of thisspecification. In the depiction of FIG. 2, the steps of the processproceed from left to right. As in O11 biosynthesis, WbpL and WbjEsynthesize the core disaccharide. Then, the S. aureusglycosyltransferase Cap5I adds D-ManNAcA. Cap5H adds an acetyl group tothe second FucNAc residue. Acetylation may be the final step of RUsynthesis as shown in FIG. 2. Flipping is possible by one or all of theWzx proteins in the system, which are recombinantly expressed Wzx of P.aeruginosa or Cap5K, or endogenously expressed Wzx-like enzymes e.g. ofthe ECA cluster encoded in the E. coli chromosome. Polymerization is anexclusive activity of the Cap5J polymerase forming the CP5polysaccharide on UndPP. As other UndPP linked polysaccharides, the CP5sugar is transferred to Lipid A core by the E. coli enzyme WaaL to formrecombinant LPS (LPS capsule).

FIG. 3 depicts the preparation of nucleotide-activated monosaccharidesin the cytoplasm by enzymes provided in the O-antigen cluster of P.aeruginosa O11, by house keeping enzymes of the Gram-negative host cell,and by S. aureus and/or E. coli enzymes known to be required forUDP-ManNAcA biosynthesis (Cap8OP and/or WecBC), as would be apparent toone of ordinary skill in the art in light of this specification. In thedepiction of FIG. 3, the steps of the process proceed from left toright. As in O11 biosynthesis, WbpL and WbjE synthesize the coredisaccharide. Then, the S. aureus glycosyltransferase Cap8H addsD-ManNAcA. Cap8J adds an acetyl group to the second FucNAc residue. Itis not known if acetylation occurs on the activated sugar or the lipidbound RU. Flipping is possible by one or all of the Wzx proteins in thesystem, which are recombinantly expressed Wzx of P. aeruginosa or Cap8K,or endogenously expressed Wzx-like enzymes e.g. of the ECA clusterencoded in the E. coli chromosome. Polymerization is an exclusiveactivity of the Cap8I polymerase forming CP8 polysaccharide on UndPP.The CP8 sugar is then transferred to Lipid A core in E. coli by theenzyme WaaL.

FIG. 4 illustrates the different structures of the O11, CP5 and CP8polysaccharides. It is shown in FIG. 4 that the RUs share the identicalstem structure consisting of the UndPP and the disaccharideα-D-FucNAc-(1,3)-L-FucNAc. The S. aureus RUs are partially decoratedwith a single O-acetyl group, either on the middle L-FucNAc or on theManNAcA residue, which is characteristic for the S. aureus RUs. Theconnectivity of the second and third sugar in the S. aureus RUs isdifferent between them, as well as the connectivity between thepolymerized RUs. On the right, the sugar structures are shown in adifferent representation. The number by the back arrows (CP5 and CP8)indicates the position of the carbon modified with an O-acetyl group. Analternative representation of the RU structures is shown on the bottomleft. As shown in FIG. 4, there is great overlap between the RU in theO11 antigen that is part of a polysaccharide native to P. aeruginosa andthose of the CP5 and CP8 capsules of the respective strains ofStaphylococcus. In particular, as show in FIG. 4, the L-FucNAc->D-FucNAcportion in the RU it is identical in both.

In another aspect, the invention features a method of identifying atarget polysaccharide for use in glycosylating a protein with saidtarget polysaccharide, in whole or in part. Said glycosylated proteincomprising the target polysaccharide can be used, for example, invaccine compositions. The method of identifying a target polysaccharideincludes: identifying a Gram-positive bacterium, such as S. aureus, as atarget; identifying a first repeating unit of a polysaccharide producedby said Gram-positive bacterium comprising at least three monomers;identifying a polysaccharide produced by a bacterium of a Gram-negativespecies comprising a second repeating unit comprising at least two ofthe same monomers as said first repeating monomer unit.

Accordingly, in one embodiment of the invention, a method of modifying abacterium of a first Gram-negative species includes: identifying aGram-positive bacterium, such as S. aureus, as a target; identifying afirst repeating unit of a polysaccharide produced by said Gram-positivebacterium comprising at least three monomers; identifying apolysaccharide produced by a bacterium of a second Gram-negative speciescomprising a second repeating unit comprising at least two of the samemonomers as said first repeating unit; inserting into said bacterium ofa first Gram-negative species one or more nucleotide sequences encodingglycosyltransferases that assemble a trisaccharide containing: a) saidsecond repeating unit; and b) a monomer of said first repeating unit notpresent in said second repeating unit; inserting a nucleotide sequenceencoding a protein, such as a protein comprising at least one insertedconsensus sequence D/E-X-N-Z-S/T, wherein X and Z may be any naturalamino acid except proline; and inserting a nucleotide sequence encodingan OTase.

In an embodiment of the invention, the method further comprisesinserting into a host Gram-negative bacterium one or more nucleotidesequences encoding glycosyltransferases that assemble a trisaccharidecontaining a monomer of a first repeating unit not present in a secondrepeating unit and that assemble the second repeating unit. Anadditional embodiment of the invention involves inserting one or moreglycosyltransferases from a Gram-negative bacterium that assemble atleast one monomer unit from a first repeating unit and one or moreglycosyltransferases from a Gram-positive bacterium, such as S. aureus,that assemble at least two monomers from a second repeating unit. Themethod additionally comprises inserting into inserting into aGram-negative host bacterium a nucleotide sequence encoding a proteinand a nucleotide sequence encoding an OTase.

In at least one embodiment of the invention, a host E. coli strain isgenerated carrying the corresponding nucleic acids encoding the requiredenzymes from the CP5 and CP8 strains of S. aureus, which will build up,flip and polymerize the constructed repeating units. In an embodiment,the specific glycosyltransferases needed correspond to those forming theL-FucNAc->D-FucNAc RU that are native to P. aeruginosa, and toglycosyltransferases corresponding to the ones adding the D-ManNAcAmonosaccharide to the complete the RU that are native to each of the CP5and CP8 strains of S. aureus. Such an embodiment may further includeusing a plasmid to inject the nucleic acids into the host cell. Anadditional embodiment involves using, in one plasmid, nucleic acidsencoding for the glycosyltransferases corresponding toL-FucNAc->D-FucNAc, and, in a different plasmid, nucleic acids encodingfor the glycosyltransferases corresponding to D-ManNAcA. One benefit ofsuch embodiments, surprising in light of the prior art, is that themodified LPS biosynthesis pathway of P. aeruginosa that is nowresponsible for producing the constructed RU polymer of the S. aureuscapsule results in a structure that is much smaller than the capsule ofS. aureus.

The instant invention is additionally directed to a recombinantN-glycosylated protein comprising at least one inserted consensussequence D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acidexcept proline; and at least one oligo- or polysaccharide from aGram-positive bacterium linked to said consensus sequence. In anotherembodiment, the recombinant N-glycosylated protein comprises two or moreof said inserted consensus sequences. In yet an additional embodiment,the recombinant N-glycosylated protein comprises two or more of said S.aureus oligo- or polysaccharides. In a still further embodiment, therecombinant N-glycosylated protein comprises two or more of saidinserted consensus sequences and oligo- or polysaccharides fromdifferent S. aureus strains, for example, from S. aureus capsularpolysaccharide 5 strain and capsular polysaccharide 8 strain.

The present invention is furthermore directed to a combination of amodified capsular polysaccharide of S. aureus with a protein antigenfrom the same organism by N-glycosidic linkage.

Embodiments of the present invention include a protein that isglycosylated in nature. Such naturally glycosylated proteins (e.g., C.jejuni proteins) contain natural consensus sequences but do not compriseany additional (i.e., introduced) optimized consensus sequences.Naturally glycosylated proteins include prokaryotic and eukaryoticproteins. Embodiments of the instant invention further include arecombinant N-glycosylated protein, comprising one or more of thefollowing N-glycosylated partial amino acid sequence(s): D/E-X-N-Z-S/T,(optimized consensus sequence) wherein X and Z may be any natural aminoacid except Pro, and wherein at least one of said N-glycosylated partialamino acid sequence(s) is introduced. The introduction of specificpartial amino acid sequence(s) (optimized consensus sequence(s)) intoproteins leads to proteins that are efficiently N-glycosylated by anOTase, such as, for example, an OTase from Campylobacter spp., such as,for example, an OTase from C. jejuni, at the positions of introduction.

The term “partial amino acid sequence(s)” as it is used in the contextof the present invention will also be referred to as “optimizedconsensus sequence(s)” or “consensus sequence(s)”. The optimizedconsensus sequence is N-glycosylated by an OTase, such as, for example,an OTase from Campylobacter spp., such as, for example, an OTase from C.jejuni.

In accordance with the internationally accepted one letter code foramino acids the abbreviations D, E, N, S and T denote aspartic acid,glutamic acid, asparagine, serine, and threonine, respectively.

The introduction of the optimized consensus sequence can be accomplishedby the addition, deletion and/or substitution of one or more aminoacids. The addition, deletion and/or substitution of one or more aminoacids for the purpose of introducing the optimized consensus sequencecan be accomplished by chemical synthetic strategies well known to thoseskilled in the art such as solid phase-assisted chemical peptidesynthesis. Alternatively, and preferred for larger polypeptides, theproteins of the present invention can be prepared by standardrecombinant techniques by adding nucleic acids encoding for one or moreoptimized consensus sequences into the nucleic acid sequence of astarting protein, which may be a protein that is naturally glycosylatedor may be a protein that is not naturally glycosylated.

In a preferred embodiment, the proteins of the present invention maycomprise one or more, preferably at least two or at least three, andmore preferably at least five of said introduced N-glycosylatedoptimized amino acid sequences.

The presence of one or more N-glycosylated optimized amino acidsequence(s) in the proteins of the present invention can be of advantagefor increasing their antigenicity, increasing their stability, affectingtheir biological activity, prolonging their biological half-life and/orsimplifying their purification.

The optimized consensus sequence may include any amino acid exceptproline in position(s) X and Z. The term “any amino acids” is meant toencompass common and rare natural amino acids as well as synthetic aminoacid derivatives and analogs that will still allow the optimizedconsensus sequence to be N-glycosylated by the OTase. Naturallyoccurring common and rare amino acids are preferred for X and Z. X and Zmay be the same or different.

It is noted that X and Z may differ for each optimized consensussequence in a protein according to the present invention.

The N-glycan bound to the optimized consensus sequence will bedetermined by the specific glycosyltransferases and their interactionwhen assembling the oligosaccharide on a lipid carrier for transfer bythe OTase. Those skilled in the art can design the N-glycan by varyingthe type(s) and amount of the specific glycosyltransferases present inthe desired host cell. (Raetz & Whitfield, LipopolysaccharideEndotoxins, NIH-PA Author Manuscript 1-57, 19-25 (published in finaledited form as: Annual Rev. Biochem., 71: 635-700 (2002)); Reeves etal., Bacterial Polysaccharide Synthesis and Gene Nomenclature, Trends inMicrobio. 4(3): 495-503, 497-98 (December 1996); and Whitfield, C. andI. S. Roberts. 1999. Structure, assembly and regulation of expression ofcapsules in Escherichia coli. Mol Microbiol 31(5): 1307-19).

“Polysaccharides” as used herein include saccharides comprising at leasttwo monosaccharides. Polysaccharides include oligosaccharides,trisaccharides, repeating units comprising one or more monosaccharides(or monomers), and other saccharides recognized as polysaccharides byone of ordinary skill in the art. N-glycans are defined herein as mono-,oligo- or polysaccharides of variable compositions that are linked to anε-amide nitrogen of an asparagine residue in a protein via anN-glycosidic linkage.

Polysaccharides of embodiments of the invention include withoutlimitation S. aureus polysaccharides such as CP5 and CP8. Embodiment ofthe invention further includes S. aureus polysaccharides that target abacterium, such as a polysaccharide that targets a methicillin-resistantstrain of S. aureus. Where it is mentioned herein that polysaccharidestarget a bacterial strain, such polysaccharides include polysaccharidesthat are from the bacterium against which an immune or antigenicresponse is desired and further include polysaccharides that are thesame as, based on, derived from, native to or engineered from thebacterium against which an immune or antigenic response is desired.

There is no limitation on the origin of the recombinant protein of theinvention. In one embodiment, said protein is derived from mammalian,bacterial, viral, fungal or plant proteins. In a further embodiment, theprotein is derived from mammalian, most preferably human proteins. Forpreparing antigenic recombinant proteins according to the invention,preferably for use as active components in vaccines, it is preferredthat the recombinant protein is derived from a bacterial, viral orfungal protein. Glycosylation of proteins of various origins is known toone of skill in the art. Kowarik et al. “Definition of the bacterialN-glycosylation site consensus sequence” EMBO J. (2006) 1-10.

In an example in an embodiment, genetically detoxified P. aeruginosaExotoxin (EPA) is a suitable protein carrier. For producing a version ofEPA that may be glycosylated, the nucleic acids encoding for EPA need tobe modified by insertion of glycosylation sites as previously discussed.

Protein carriers intended for use in embodiments of the invention shouldpreferably have certain immunological and pharmacological features. Froman immunological perspective, preferably, a protein carrier should: (1)have T-cell epitopes; (2) be capable of delivering an antigen to antigenpresenting cells (APCs) in the immune system; (3) be potent and durable;and (4) be capable of generating an antigen-specific systemic IgGresponse. From a pharmacological perspective, a protein carrier shouldpreferably: (1) be non-toxic; and (2) be capable of delivering antigensefficiently across intact epithelial barriers. More preferably, inaddition to these immunological and pharmacological features, a proteincarrier considered for use in the production of a bacterial bioconjugateshould: (1) be easily secreted into the periplasmic space; and (2) becapable of having antigen epitopes readily introduced as loops or linearsequences into it. Informed by this disclosure and knowledge of one ofordinary skill in the art, a practitioner of ordinary skill in the artmay routinely consider and identify suitable protein carriers that maybe used in particular embodiments of the invention.

In an embodiment of the invention, the Campylobacter protein AcrA is aprotein carrier.

In a further embodiment of the invention, genetically detoxified P.aeruginosa Exotoxin (EPA) is a protein carrier in which the targetorganism for which a vaccine is desired is S. aureus. Unlike AcrA whichcontains natural glycosylation sites, EPA contains no such naturalglycosylation sites and needs to be modified by insertion ofglycosylation sites (e.g., insertion of nucleic acids encoding for theoptimized consensus sequence as discussed earlier into the nucleic acidsequence encoding for EPA). In an additional embodiment, EPA is modifiedto introduce two glycosylation sites that will allow glycosylation withthe S. aureus antigen. In a still further embodiment, two consensussequences are introduced as discussed in Example 10 of WO 2009/104074.

The amino acid sequence of EPA, as modified in an embodiment of thisinvention to contain two glycosylation sites, is provided as SEQ ID NO:13 (with signal sequence) and SEQ ID NO.: 14 (without signal sequence).The glycosylation sites in SEQ ID NO: 13 are DNNNS and DQNRT atpositions 260DNNNS and 402DQNRT. The glycosylation sites in SEQ ID NO:14 are DNNNS and DQNRT at positions 241DNNNS and 383DQNRT.

A carrier protein such as EPA is a protein on which N-glycosylationsites may be added in the production of a bacterial bioconjugate.N-glycosylation sites require introduction of the consensus sequencesdiscussed previously, namely, insertion of D/E-X-N-Z-S/T sequons,wherein X and Z may be any natural amino acid except proline. We havefound that such consensus sequences preferably are introduced in surfaceloops, by insertion rather than mutation and by the use of additionallyinserted flanking residues and by mutation of flanking residues tooptimize the operation of the N-glycosylation site.

Some well-characterized protein subunit antigens of S. aureus are thealpha hemolysin (alpha toxin, Hla), clumping factor alpha (ClfA), IsdB,and Panton-Valentine Leukocidin (PVL).

Hla is a secreted pore-forming toxin and an essential virulence factorof MRSA in a mouse model of S. aureus pneumonia. The level of Hlaexpression by independent S. aureus strains directly correlates withtheir virulence. Active immunization with a mutant form of Hla (HlaH35L, SEQ ID NO: 5), which cannot form pores (Menzies, B. E., and D. S.Kernodle. 1996. Passive immunization with antiserum to a nontoxicalpha-toxin mutant from Staphylococcus aureus is protective in a murinemodel. Infect Immun 64:1839-41; Jursch, R., A. Hildebrand, G. Hobom, J.Tranum-Jensen, R. Ward, M. Kehoe and S. Bhakdi. 1994. Histidine residuesnear the N terminus of staphylococcal alpha-toxin as reporters ofregions that are critical for oligomerization and pore formation. InfectImmun 62(6): 2249-56), was shown to generate antigen-specificimmunoglobulin G responses and to afford protection againststaphylococcal pneumonia. Transfer of Hla-specific antibodies protectsnaive animals against S. aureus challenge and prevents the injury ofhuman lung epithelial cells during infection (Bubeck Wardenburg, J., A.M. Palazzolo-Ballance, M. Otto, O. Schneewind, and F. R. DeLeo. 2008.Panton-Valentine leukocidin is not a virulence determinant in murinemodels of community-associated methicillin-resistant Staphylococcusaureus disease. J Infect Dis 198:1166-70). To be used as a vaccine, theH35L mutation in Hla is required to eliminate toxicity of the protein(Menzies, B. E., and D. S. Kernodle. 1994. Site-directed mutagenesis ofthe alpha-toxin gene of Staphylococcus aureus: role of histidines intoxin activity in vitro and in a murine model. Infect Immun 62:1843-7).ClfA contains a protease resistant domain which is used forimmunization. Passive immunization of mice with anti-ClfA and anti CP5antibodies effectively sterilized mammary glands in mammary glandinfection model (Tuchscherr, L. P., F. R. Buzzola, L. P. Alvarez, J. C.Lee, and D. O. Sordelli. 2008. Antibodies to capsular polysaccharide andclumping factor A prevent mastitis and the emergence of unencapsulatedand small-colony variants of Staphylococcus aureus in mice. Infect Immun76: 5738-44).

A further embodiment of the invention includes glycosylation of proteinsnative to S. aureus, for example, Hla and ClfA. In additional exampleembodiments of the invention, the protein carrier used may be selectedto be the Hla protein, for example Hla H35L (for example, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 16). In another additionalexample embodiment of the invention, the protein carrier is the ClfAprotein (for example, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12).

The invention is further directed to recombinant host prokaryoticorganisms comprising: a nucleotide sequence encoding one or moreglycosyltransferase of a first prokaryotic species, such as aGram-positive species; one or more glycosyltransferases of a differentprokaryotic species, such as a Gram-negative species; a nucleotidesequence encoding a protein; and a nucleotide sequence encoding anOTase. The invention is additionally directed to a recombinant hostprokaryotic organism comprising an introduced nucleotide sequenceencoding glycosyltransferases native only to a Gram-positive prokaryoticorganism; a nucleotide sequence encoding a protein; and a nucleotidesequence encoding an OTase. The invention is also directed to arecombinant or engineered host prokaryotic organism comprising: anucleotide sequence encoding a glycosyltransferase native to a firstprokaryotic species, which is, for example, different from the hostprokaryotic organism; a nucleotide sequence encoding aglycosyltransferase native to a second prokaryotic species differentfrom the species of said first prokaryotic organism and, for example,different from said host. The engineered prokaryotic organism can also,for example, comprise a first prokaryotic species that is aGram-positive species. The engineered prokaryotic organism can also, forexample, comprise a second prokaryotic species that is a Gram-negativespecies. The invention further includes a recombinant or engineeredGram-negative host prokaryotic organism comprising: a nucleotidesequence encoding a glycosyltransferase native to a Gram-negativeprokaryotic species that is, for example, different from said hostprokaryotic organism; a nucleotide sequence encoding aglycosyltransferase native to S. aureus; a nucleotide sequence encodinga protein; and a nucleotide sequence encoding an OTase. The inventionfurther includes a recombinant or engineered E. coli host comprising: anucleotide sequence encoding a glycosyltransferase native to P.aeruginosa; a nucleotide sequence encoding one or moreglycosyltransferases native to S. aureus CP5 strain and/or to S. aureusCP8 strain; a nucleotide sequence encoding a P. aeruginosa EPA, S.aureus alpha hemolysin, or S. aureus clumping factor A protein carrier;and a nucleotide sequence encoding an OTase, for example, and OTasenative to C. jejuni.

In addition to using the biosynthesis pathway of the other Gram-negativeorganism in the modified host E. coli organism, in a further embodiment,also included within the host E. coli organism are nucleic acidsencoding for (i) glycosyltransferases for construction the structure ofthe repeating units of the polysaccharide of the other Gram-negativeorganism (that are identical to the repeating units of thepolysaccharide of interest of the target Gram-positive S. aureusorganism), and (ii) glycosyltransferases for construction of the unitsof the polysaccharide of interest of the target Gram-positive S. aureusorganism that are not found in the relevant polysaccharide of the otherGram-negative organism, and (iii) enzymes for flipping andpolymerization of the constructed RU of interest of the targetGram-positive S. aureus organism to form a S. aureus capsule likepolysaccharide. In particular, in this embodiment, the nucleic acidsencoding for (i) originated with the other Gram-negative bacterium,whereas the nucleic acids encoding for (ii) and (iii) originated withthe target Gram-positive S. aureus organism.

Another aspect of the invention is directed to: an engineered hostprokaryotic organism comprising: i) a nucleotide sequence encodingglycosyltransferases native to a Gram-positive prokaryotic species; ii)a nucleotide sequence encoding a protein; and iii) a nucleotide sequenceencoding an OTase, wherein the sequences encoding transporter genes ofsaid Gram-positive prokaryotic species are deleted. Such an embodimentinvolves an introduced nucleic acid construct that encodes onlyGram-positive glycosyltransferases.

Regarding the other nucleic acids that would be inserted into the hostin one or more other embodiments, nucleic acids encoding a protein, suchas AcrA, Hla, ClfA or EPA (SEQ ID NO: 15, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 16; SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12; SEQ ID NO: 13, SEQ ID NO: 14), and the oligosaccharyltransferase ofC. jejuni (SEQ ID NO: 27), which are part of the glycosylation machineryof that organism, are injected into the host in addition to the nucleicacids encoding for glycosyltransferases from each of P. aeruginosa andS. aureus. As a result, the modified E. coli organism can glycosylatethe AcrA protein with the polysaccharide produced in that organism byaction of the glycosyltransferases from S. aureus and the otherGram-negative bacterium.

One embodiment of the invention involves an engineered host prokaryoticorganism comprising: i) a nucleotide sequence encoding aglycosyltransferase native to a first prokaryotic species different fromthe host prokaryotic organism; ii) a nucleotide sequence encoding aglycosyltransferase native to a second prokaryotic species, for example,a Gram-positive prokaryotic species, different from the host prokaryoticorganism; iii) a nucleotide sequence encoding a protein; and iv) anucleotide sequence encoding an OTase. In embodiments of the invention,the first prokaryotic species is a Gram-negative species, for example,P. aeruginosa.

In the context of the present invention, host cells refer to any hostcell, e.g., an eukaryotic or prokaryotic host cell. In other embodimentsthe host cell is a prokaryotic host cell, e.g. Escherichia ssp.,Campylobacter ssp., Salmonella ssp., Shigella ssp., Helicobacter ssp.,Pseudomonas ssp. or Bacillus ssp. In still further embodiments, the hostcell is Escherichia coli, Campylobacter jejuni, Salmonella typhimurium,etc.

The invention is furthermore directed to methods of producing abioconjugate vaccine comprising introducing into a host prokaryoticorganism nucleic acids encoding one or more glycosyltransferases of S.aureus; one or more glycosyltransferases of a second prokaryoticspecies, a protein; and an OTase. In addition, the present invention isdirected to the production of bioconjugate vaccines by producing inGram-negative bacteria modified capsular polysaccharides on undecaprenol(Und), and linking these polysaccharide antigens to a protein carrier ofchoice.

The invention is further directed to methods of producing glycosylatedproteins in a host prokaryotic organism comprising nucleotide sequenceencoding glycosyltransferases native to a first prokaryotic organism andalso encoding glycosyltransferases native to a second prokaryoticorganism that is different from the first prokaryotic organism. Thepresent invention is additionally directed to the production of proteinsN-glycosylated with capsular polysaccharides of Gram-positive bacteria,which are synthesized by a combination of different glycosyltransferasesfrom different organisms. The invention is furthermore directed to theproduction of glycosylated proteins in a host prokaryotic organismcomprising an introduced nucleotide sequence encodingglycosyltransferases native only to a Gram-positive prokaryoticorganism.

As in known in the art, the biosynthesis of different polysaccharides isconserved in bacterial cells. The polysaccharides are assembled oncarrier lipids from common precursors (activated sugar nucleotides) atthe cytoplasmic membrane by different glycosyltransferases with definedspecificity. (Whitfield, C., and I. S. Roberts. 1999. Structure,assembly and regulation of expression of capsules in Escherichia coli.Mol Microbiol 31: 1307-19). The biosynthetic pathway for polysaccharideproduction of O-antigen in Gram-negative and for capsular polysaccharideType I in Gram-positive is conserved. The process uses the same lipidcarrier, i.e., UndP, for polysaccharide assembly. It starts with theaddition of a monosaccharide-1-phosphate to the carrier lipid UndP atthe cytoplasmic side of the membrane. The antigen is built up bysequential addition of monosaccharides from activated sugar nucleotidesby different glycosyltransferases. The lipid-linked oligosaccharide orRU is then flipped through the membrane by the flippase. RUs arepolymerized by the enzyme Wzy in the periplasmic space, forming theso-called O-antigen in Gram negative bacteria or capsular polysaccharidein Gram-positive bacteria. Gram negative bacteria use the Wzz enzyme toregulate the length of the polymer, which is then transferred to lipid Acore forming LPS. LPS is further translocated to the outer membraneexposing the O-antigen to the outside (as depicted, for example, in FIG.1). Gram-positive bacteria, in contrast, form the capsule from thislipid-bound precursor by further transport using a different andspecialized enzymatic machinery. The biosynthetic pathways of thesepolysaccharides enable the production of bioconjugates in vivo bycapturing the polysaccharides in the periplasm onto a protein carrier.

The process of polysaccharide construction differs for capsularpolysaccharides in that the capsular polysaccharide is released from thecarrier lipid after polymerization and exported to the surface. InGram-positive bacteria like S. aureus that do not contain a periplasmiccompartment, the polymerization of the antigen takes place at the outerside of the membrane. In addition, length regulation in S. aureus isincluded in the machinery of three enzymes responsible for capsuleassembly. In this assembly, the polysaccharide is released from thecarrier lipid and exported to the surface by an enzymatic process.

The genetic elements found in the gene cluster required for functionalcapsule expression in S. aureus resemble the genetic machinery found inwzy dependent O-antigen synthesis clusters. (Dean, C. R., C. V.Franklund, J. D. Retief, M. J. Coyne, Jr., K. Hatano, D. J. Evans, G. B.Pier, and J. B. Goldberg. 1999. Characterization of the serogroup O11O-antigen locus of Pseudomonas aeruginosa PA103. J Bacteriol181:4275-4284).

Despite these differences between polysaccharide construction inGram-positive and Gram-negative bacteria, it was surprisingly discoveredand verified that aspects of the LPS pathway in a Gram-negative organismcould be used to produce polysaccharides that contain some of the samerepeating units as capsular polysaccharides native to Gram-positivebacteria, such as, for example, S. aureus. As such polysaccharides areproduced by LPS pathway mechanisms in the Gram-negative host, thestructure of such polysaccharides is the same as in LPS polysaccharideprecursors. Such polysaccharides produced in Gram-negative systems ofthe instant invention can be characterized, therefore, as “modifiedcapsular polysaccharides” or “LPS capsules” for purposes of thisapplication. Furthermore, this newly synthesized expression system andbiosynthetic pathway, which combines the LPS and capsular biosyntheticpathways, may be characterized as being a “modified LPS biosyntheticpathway” for purposes of this application.

In one embodiment of the present invention, a modified polysaccharideproduced by a modified LPS biosynthetic pathway comprises:

${\underset{1,4}{\overset{\beta}{}}\begin{pmatrix}\underset{\downarrow}{3\; {Ac}} \\{D\text{-}{{ManNAcA}\underset{1,4}{\overset{\beta}{}}L}\text{-}{{FucNAc}\underset{1,4}{\overset{\alpha}{}}D}\text{-}{FucNAc}}\end{pmatrix}_{n}}\underset{1}{\rightarrow}.$

In a further embodiment of the present invention, a modifiedpolysaccharide produced by a modified LPS biosynthetic pathwaycomprises:

${\underset{1,3}{\overset{\alpha}{}}\begin{pmatrix}\underset{\downarrow}{4\; {Ac}} \\{D\text{-}{{ManNAcA}\underset{1,3}{\overset{\beta}{}}L}\text{-}{{FucNAc}\underset{1,3}{\overset{\alpha}{}}D}\text{-}{FucNAc}}\end{pmatrix}_{n}}\underset{1}{\rightarrow}.$

Using the technology of the invention, bacterial bioconjugates can beproduced that are immunogenic. Genetic modifications can be madeallowing in vivo conjugation of bacterial polysaccharides in desiredproteins and at desired positions.

Another aspect of the invention involves production of LPS-capsules ormodified LPSs conjugated to a protein carrier using the modified LPSbiosynthetic pathway as discussed above.

A further embodiment of the invention includes a nucleotide sequenceconstruct that encodes the Cap5 and Cap8 complete polysaccharidebiosynthesis cluster, wherein the deleted transporter genes are capA,capB and capC of S. aureus (see FIG. 6).

An additional embodiment of the invention includes integrating theCP5/O11 chimeric cluster (SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 17)or the CP8/O11 chimeric cluster (SEQ ID NO. 4, SEQ ID NO. 18 or SEQ IDNO. 19) into the genome of a host cell. A further embodiment of theinvention involves integrating into the genome of a host cell: (a) theCP5/O11 chimeric cluster (SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 17)or CP8/O11 chimeric cluster (SEQ ID NO. 4 SEQ ID NO. 18 or SEQ ID NO.19); (b) nucleic acids encoding the OTase; and (c) nucleic acidsencoding a protein with or without an introduced consensus sequence.

Another embodiment of the instant invention is directed to plasmids,such as, for example, plasmids comprising one or more of SEQ. ID NO: 2;SEQ. ID NO: 3; SEQ ID NO: 4; SEQ. ID NO: 17; SEQ. ID NO: 18 and SEQ. IDNO: 19. The invention also includes plasmids comprising one or more ofSEQ. ID NO: 13; SEQ. ID NO: 14 and SEQ. ID NO: 15. The invention alsorelates to plasmids comprising one or more of SEQ ID NO: 16; SEQ. ID NO:6; SEQ. ID NO: 7 and SEQ. ID NO: 8. The invention also relates toplasmids comprising one or more of SEQ ID NO: 10; SEQ. ID NO: 11 andSEQ. ID NO: 12. Moreover, the invention is directed to plasmidscomprising one or more of SEQ. ID NO: 20; SEQ. ID NO: 21 and SEQ. ID NO:27.

Embodiments of the instant invention furthermore are directed totransformed bacterial cells, such as, for example, including a bacterialcell transformed with a plasmid comprising one or more of SEQ. ID NO. 2;SEQ. ID NO. 3; SEQ. ID NO: 4; SEQ. ID NO: 17; SEQ. ID NO: 18; SEQ. IDNO: 19; SEQ. ID NO: 20; SEQ. ID NO: 21 and SEQ. ID NO: 27. Furtherincluded in the invention is a bacterial cell transformed with a plasmidcomprising one or more of SEQ. ID NO: 19 and SEQ ID NO: 20. Additionallyincluded is a bacterial cell transformed with a plasmid comprising oneor more of SEQ ID NO: 13, SEQ ID NO: 19 and SEQ ID NO: 21. The instantinvention is further directed to a bacterial cell transformed with aplasmid comprising one or more of SEQ. ID NO: 16, SEQ ID NO: 6; SEQ IDNO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12. Theinvention is additionally directed to transformed bacterial cells, suchas, for example, including a bacterial cell transformed with a plasmidcomprising one or more of SEQ. ID NO. 3; SEQ. ID NO: 4; SEQ. ID NO: 17;SEQ. ID NO: 18; and SEQ. ID NO: 19, and wherein said bacterial cellexpresses a glycosyltransferase native to P. aeruginosa and aglycosyltransferase native to S. aureus CP5 and/or CP8. Further includedin the invention is a bacterial cell transformed with a plasmidcomprising one or more of SEQ. ID NO: 17; SEQ ID NO: 18 and SEQ. ID NO:19 wherein said bacterial cell expresses a glycosyltransferase native toP. aeruginosa, a glycosyltransferase native to S. aureus CP5 and/or CP8and PglB. Still further included in the instant invention is (a) abacterial cell transformed with a plasmid comprising SEQ. ID NO. 19,wherein said bacterial cell expresses a glycosyltransferase native to P.aeruginosa, a glycosyltransferase native to S. aureus CP8, Wzz of E.coli serovar O7 and PglB; (b) a bacterial cell transformed with aplasmid comprising one or more of SEQ. ID NO. 19 and SEQ. ID NO. 20,wherein said bacterial cell expresses a glycosyltransferase native to P.aeruginosa, a glycosyltransferase native to S. aureus CP8, Wzz (lengthregulator), EPA and PglB; and (c) a bacterial cell comprising one ormore of SEQ. ID NO. 16; SEQ. ID NO: 6; SEQ. ID NO: 7; SEQ. ID NO: 8;SEQ. ID NO. 13; SEQ. ID NO: 14; SEQ. ID NO: 15; SEQ. ID NO: 10; SEQ. IDNO: 11 and SEQ. ID NO: 12.

Embodiments of the instant invention are additionally directed to amethod of inducing an immune response against an infection caused byGram-positive and other bacteria in a mammal, such as, for example, in ahuman. In one embodiment, the method comprises administering to saidmammal an effective amount of a pharmaceutical composition comprising:protein comprising at least one inserted consensus sequenceD/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid exceptproline; and one or more oligo- or polysaccharides, the one or moreoligo- or polysaccharides being the same or different as another of theone or more oligo- or polysaccharides, from a Gram-positive bacteriumlinked to said consensus sequence. A further embodiment of the presentinvention includes a method of inducing an immune response against aninfection caused by S. aureus in a mammal, comprising administering tosaid mammal an effective amount of a pharmaceutical compositioncomprising: an inserted consensus sequence D/E-X-N-Z-S/T, wherein X andZ may be any natural amino acid except proline; at least one S. aureusoligo- or polysaccharide, such as CP5 polysaccharide; and apharmaceutically acceptable adjuvant. Another embodiment of theinvention is directed to inducing an immune response against aninfection caused by S. aureus in a mammal, comprising administering tosaid mammal an effective amount of a pharmaceutical compositioncomprising: a protein comprising an inserted consensus sequenceD/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid exceptproline; at least one S. aureus CP8 polysaccharide; and apharmaceutically acceptable adjuvant. A still further embodiment isdirected to inducing an immune response against an infection caused byS. aureus in a mammal, comprising administering an effective amount of apharmaceutical composition comprising a protein with two or moreconsensus sequences and oligo- or polysaccharides from differentGram-positive bacterial strains. A still further embodiment is directedto inducing an immune response against an infection caused by S. aureusin a mammal, comprising administering an effective amount of apharmaceutical composition comprising a protein with two or moreconsensus sequences and polysaccharides comprising S. aureus CP5 and S.aureus CP8.

In instances in this specification where specific nucleotide or aminoacid sequences are noted, it will be understood that the presentinvention encompasses homologous sequences that still embody the samefunctionality as the noted sequences. In an embodiment of the invention,such sequences are at least 85% homologous. In another embodiment, suchsequences are at least 90% homologous. In still further embodiments,such sequences are at least 95% homologous. The determination of percentidentity between two nucleotide or amino acid sequences is known to oneof skill in the art.

Nucleic acid sequences described herein, such as those described in thesequence listings accompanying this specification, are examples only,and it will be apparent to one of skill in the art that these sequencescan be combined in different ways. Additional embodiments of theinvention include variants of nucleic acids. A variant of a nucleic acid(e.g., a codon-optimized nucleic acid) can be substantially identical,that is, at least 70% identical, for example, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical, to SEQ IDNO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,SEQ ID NO: 26 and/or SEQ ID NO: 27. Nucleic acid variants of a sequencethat contains SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO. 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and/or SEQ ID NO: 27. Includenucleic acids with a substitution, variation, modification, replacement,deletion, and/or addition of one or more nucleotides (e.g., 2, 3, 4, 5,6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200,250, 300, 350, 400, 450, 500 or more nucleotides) from a sequence thatcontains SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID. NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:24, SEQ ID NO: 25, SEQ ID NO: 26 and/or SEQ ID NO: 27, or parts thereof.

Such variants include nucleic acids that encode prokaryoticglycosyltransferases and that i) are expressed in a host cell such as E.coli and ii) are substantially identical to SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 17, SEQ ID NO: 18 and/or SEQ ID NO: 19 and/orparts thereof.

Nucleic acids described herein include recombinant DNA and synthetic(e.g., chemically synthesized) DNA. Nucleic acids can be double-strandedor single-stranded. In the case of single-stranded nucleic acids, thenucleic acid can be a sense strand or antisense strand. Nucleic acidscan be synthesized using oligonucleotide analogs or derivatives, asknown to one of skill in the art in light of this specification.

Plasmids that include a nucleic acid described herein can be transformedinto host cells for expression. Techniques for transformation are knownto those of skill in the art in light of this specification.

An additional embodiment of the invention involves producingGram-positive bioconjugate vaccines containing LPS-capsules or modifiedLPSs conjugated to a protein carrier.

A further embodiment of the invention involves a novel bioconjugatevaccine. A further embodiment of the invention involves a novel approachfor producing such bioconjugate vaccines that uses recombinant bacterialcells that directly produce immunogenic or antigenic bioconjugates. Inone embodiment, bioconjugate vaccines can be used to treat or preventbacterial diseases, such as diarrhea, nosocomial infections andmeningitis. In further embodiments, bioconjugate vaccines may havetherapeutic and/or prophylactic potential for cancer or other diseases.

In another embodiment of the present invention synthesized complexes ofpolysaccharides (i.e., sugar residues) and proteins (i.e., proteincarriers) can be used as conjugate vaccines to protect againstinfections such as S. aureus infections. In one embodiment, abioconjugate vaccine, such as a Gram-positive vaccine, comprises aprotein carrier comprising an inserted nucleic acid consensus sequence;at least one oligo- or polysaccharide from a Gram-positive bacteriumlinked to the consensus sequence, and, optionally, an adjuvant. Thepresent invention is further directed in another embodiment to aGram-positive bioconjugate vaccine, such as a S. aureus vaccine,comprising a protein carrier comprising an inserted nucleic acidconsensus sequence; at least one oligo- or polysaccharide from aGram-positive bacterium, such as capsular polysaccharide or LPS capsule,linked to the consensus sequence, and, optionally, an adjuvant. Inanother embodiment of the invention, the S. aureus bioconjugate vaccinecomprises two or more of these inserted consensus sequences. In afurther embodiment, the S. aureus bioconjugate vaccine comprises two ormore of S. aureus oligo- or polysaccharides. A still further embodimentcomprises two or more of said inserted consensus sequences and oligo- orpolysaccharides from different S. aureus strains, for example, from S.aureus capsular polysaccharide 5 strain (CP5) and capsularpolysaccharide 8 strain (CP8).

An additional embodiment of the present invention involves an S. aureusvaccine made by a glycosylation system using a modified LPS pathway,which comprises the production of a modified capsular polysaccharide orLPS-capsule. A further additional embodiment involves an S. aureusvaccine made by a glycosylation system using a modified LPS pathway,which comprises the production of a modified capsular polysaccharidefrom introduced nucleic acids that do not encode glycosyltransferases ofa Gram-negative prokaryotic species.

A further embodiment involves an S. aureus vaccine produced by aglycosylation system comprising nucleic acids encoding: i) one or moreglycosyltransferases responsible for producing the L-FucNAc->D-FucNAc ofthe RU of the O11 antigen native to P. aeruginosa; ii) one or moreglycosyltransferases responsible for producing the D-ManNAcA containingRU native to either the CP5 or CP8 strains of S. aureus; iii) one ormore enzymes responsible for flipping and polymerization of the CP5 orCP8 constructed RUs, iv) a recombinant protein containing introducedconsensus sequences; and v) oligosaccharyltransferase from C. jejuni. Inthis embodiment, the host organism may be a Gram-negative bacterium, forexample, E. coli.

An additional embodiment of the invention involves an S. aureus vaccineproduced by a glycosylation system comprising nucleic acids encoding: i)glycosyltransferases responsible for producing the L-FucNAc->D-FucNAc ofthe RU of the O11 antigen native to P. aeruginosa; ii) aglycosyltransferase responsible for producing the D-ManNAcA containingRU native to either the CP5 or CP8 strains of S. aureus; iii) AcrAprotein of C. jejuni; and iv) oligosaccharyltransferase from C. jejuni.In this embodiment, the host organism may be a Gram-negative bacterium,for example, E. coli.

The vaccines of the instant invention have therapeutic and prophylacticutilities. It will be appreciated that the vaccine of the invention maybe useful in the fields of human medicine and veterinary medicine. Thus,the subject to be immunized may be a human or other animal, for example,farm animals including cows, sheep, pigs, horses, goats and poultry(e.g., chickens, turkeys, ducks and geese) and companion animals such asdogs and cats.

In another aspect, the invention is directed to a method of generatingvaccines for immunizing a mammal against a bacterium such as aGram-positive bacterium. The method includes: immunizing a subject witha bioconjugate, such as a bioconjugate comprising a Gram-positivepolysaccharide, e.g., an S. aureus polysaccharide, and apharmaceutically acceptable carrier.

This invention also features vaccine compositions for protection againstinfection by a gram-positive bacterium such as S. aureus or fortreatment of gram-positive infection such as S. aureus infection. In oneembodiment, the vaccine compositions comprise one or more immunogeniccomponents such as a polysaccharide, or a fragment or portion thereof,from S. aureus. In a further embodiment, the vaccine compositionscomprise one or more immunogenic components such as a protein, or afragment or portion thereof, from a Gram-negative or Gram-positivebacterium.

One aspect of the invention provides a vaccine composition forprotection against infection by S. aureus which contains at least oneimmunogenic component or fragment of an S. aureus polysaccharide and apharmaceutically acceptable carrier. Such immunogenic components orfragments can include, for example, an S. aureus polysaccharide of atleast about two monomers in length or at least about three monomers inlength. In a further aspect of the invention, an S. aureus RU comprisessaid monomers. Such repeating units can include, for example, an S.aureus RU of at least 1 (one) in length.

Immunogenic components or fragments of the invention can be obtained,for example, by screening polysaccharides or polypeptides producedrecombinantly or through chemical synthesis, or, for example, byscreening the bioconjugate comprising a polysaccharide and a protein.Screening immunogenic components or fragments of the invention can beperformed using one or more of several different assays. For example,screening assays include ELISA and other assays known to one of ordinaryskill in the art.

In one embodiment, immunogenic components or fragments are identified bythe ability of the polysaccharide and/or protein to stimulate IgGantibodies against Gram-positive bacteria, such as S. aureus CP5 or CP8polysaccharides, as determined by, for example, the immune responseobtained in mice (FIG. 15A) and in rabbit (FIG. 15B) measuring specificanti CP5 antibodies (quantified by ELISA) against the glycoconjugatevaccine candidate CP5-EPA and other means known to a person of ordinaryskill in the art.

In one embodiment, immunogenic components or fragments are identified bythe ability of the polysaccharide and/or protein to stimulate opsonicactivity, such as opsonophagocytic killing, as determined by, forexample by the S. aureus killing (“in vitro” activity) with rabbit antiCP5-EPA antibodies (obtained in Example 7 below, see FIG. 15B) and othermeans known to a person of ordinary skill in the art.

In yet a further embodiment, immunogenic components or fragments areidentified by the ability of the polysaccharide and/or protein tostimulate humoral and/or cell-mediated immunity against Gram-positivebacteria, such as S. aureus, as determined by, for example, byprotection against bacterial infection (“challenge”) using activeimmunization in mice (FIG. 18) with CP5-EPA and other means known to aperson of ordinary skill in the art.

In an embodiment of the instant invention, a vaccine composition of theinvention can be based on a glycoprotein comprising an immunogeniccomponent or fragment of an S. aureus polysaccharide of the inventionand optionally further comprising a pharmaceutically acceptable carrieror adjuvant. In further embodiments of the instant invention, a vaccinecomposition can be based on a glycoprotein comprising an immunogeniccomponent or fragment of an S. aureus protein of the invention andoptionally further comprising a pharmaceutically acceptable carrier oradjuvant. In yet a further aspect of the invention, a vaccinecomposition can be based on a glycoprotein comprising a immunogeniccomponent or fragment of a P. aeruginosa protein of the invention andoptionally further comprising a pharmaceutically acceptable carrierand/or adjuvant.

It is well-known to those of ordinary skill in the art how to modify avaccine for administration to one mammal type, for example, mice, foradministration to another mammal type, for example, humans. For example,one of skill would readily know that deletion of the histidine tag fromthe protein carrier of a glycoprotein used in a vaccine composition inmice would render the glycoprotein suitable for administration in avaccine composition in humans. For example, deletion of the HISTIDINEtag (HIS-tag) in protein carriers such as, e.g. EPA (SEQ ID NO: 13),ClfA (SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12), and Hla (SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16) would be recognized forits use in a glycoprotein for administration to a human.

It should be understood that amelioration of any of the symptoms of aGram-positive, for example S. aureus, or other bacterial infection ordisease is a desirable clinical goal, including a lessening of thedosage of medication used for the Gram-positive-caused infection ordisease, for example an S. aureus-caused infection or disease, or otherbacterial-caused infection or disease, or an increase in the productionof antibodies in the serum or mucous of patients. It will be apparent tothose skilled in the art that some of the vaccine compositions of theinvention are useful for preventing a Gram-positive infection, forexample an S. aureus infection, or other bacterial infection, some areuseful for treating a Gram-positive infection, for example an S. aureusinfection, or other bacterial infection, and some are useful for bothpreventing and treating such infections.

Embodiments of the present invention such as vaccines and otherpharmaceutical agents optionally may be prepared using suitable andpharmaceutically acceptable carriers, excipients, diluents and/oradjuvants, as are well-known in the art and apparent in light of thisspecification. An excipient, diluent or adjuvant may be a solid,semi-solid or liquid material which may serve as a vehicle or medium forthe active ingredient. In light of this specification, one of ordinaryskill in the art in the field of preparing compositions can readilyselect the proper form and mode of administration depending upon theparticular characteristics of the product selected, the disease orcondition to be treated, the stage of the disease or condition, andother relevant circumstances (Remington s Pharmaceutical Sciences, MackPublishing Co. (1990)). The proportion and nature of thepharmaceutically acceptable diluent, excipient or adjuvant aredetermined by the solubility and chemical properties of thepharmaceutically active compound selected the chosen route ofadministration and standard pharmaceutical practice.

Accordingly, in embodiments of the invention, vaccine compositionscomprise immunogenic components or fragments, e.g., S. aureuspolysaccharide or fragment thereof and/or S. aureus or P. aeruginosaprotein or fragment thereof and optionally include a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”refers to a carrier that is non-toxic. Suitable pharmaceuticallyacceptable carriers include, for example, one or more of water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. Pharmaceutically acceptable carriers mayfurther comprise minor amounts of auxiliary substances such as wettingor emulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the antibody. Such pharmaceutically acceptablecarriers include, for example, liquid, semisolid, or solid diluents thatserve as pharmaceutical vehicles, excipients, or media. Any diluentknown in the art may be used. Exemplary diluents include, but are notlimited to, polyoxyethylene sorbitan monolaurate, magnesium stearate,methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose,sucrose, dextrose, sorbitol, mannitol, gum acacia, calcium phosphate,mineral oil, cocoa butter, and oil of theobroma.

Further, in additional embodiments of the invention, the vaccinecomposition can optionally include an adjuvant or a combination ofadjuvants, including, but not limited to particulate adjuvants such asaluminium salts (aluminium hydroxide, aluminium phosphate, aluminiumhydroxyphosphate sulphate, etc.); emulsions such as oil in water (MF59,AS03); lipid and salt combinations such as AS04; water in oil(Montanide); ISCOMS, liposomes/virosomes; nano- and microparticles,etc.; non particulated such as peptides; saponins (QS21); MPL A;cytokins; DNA derivates; bacterial toxins; etc. A further embodimentincludes adjuvants used in animals such as Freund's Complete Adjuvantand Freund's Incomplete Adjuvant, mycolate-based adjuvants (e.g.,trehalose dimycolate), bacterial lipopolysaccharide (LPS),peptidoglycans (i.e., mureins, mucopeptides, or glycoproteins such asN-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans,streptococcal preparations (e.g., OK432), DEAE-dextran, neutral oils(such as miglyol), vegetable oils (such as arachis oil), Pluronic, theRibi adjuvant system or interleukins, particularly those that stimulatecell-mediated immunity. The adjuvant used will depend, in part, on thecomposition and type of the glycoconjugate vaccine. The amount ofadjuvant to administer will depend on the type and size of mammal.Optimal dosages may be readily determined by routine methods.

A further aspect of the present invention relates to a pharmaceuticalcomposition, comprising at least one glycoprotein according to theinvention. The preparation of medicaments comprising glycoproteins iswell-known in the art. The preparation scheme for the finalpharmaceutical composition and the mode and details of itsadministration will depend on the protein, the host cell, the nucleicacid and/or the vector employed.

It will be apparent to those of skill in the art that thetherapeutically effective amount of polysaccharide or glycoprotein ofthis invention will depend, inter alia, upon the administrationschedule, the unit dose of antibody administered, whether thepolysaccharide or glycoprotein is administered in combination with othertherapeutic agents, the immune status and health of the patient, and thetherapeutic activity of the particular polysaccharide or glycoprotein.

The vaccine compositions and/or pharmaceutical preparations of theinvention may be adapted for oral, parenteral or topical use and may beadministered to the patient in the form of tablets, capsules,suppositories, solution, suspensions or any other suitable means ordosage form. In further aspects of the invention, the vaccinecompositions and/or pharmaceutical preparations may be introduced intothe subject to be immunized by any known method including, e.g., byintravenous, intradermal, intramuscular, intramammary, intraperitoneal,or subcutaneous injection; or by oral, sublingual, nasal, anal, orvaginal, delivery. The pharmaceutically active compounds of the presentinvention, while effective themselves, can be formulated andadministered in the form of their pharmaceutically acceptable salts,such as acid addition salts or base addition salts, for purposes ofstability, convenience of crystallization, increased solubility, and thelike. Vaccine compositions in an embodiment of the invention areadministered parenterally, e.g., by injection, either subcutaneously orintramuscularly. Methods for intramuscular immunization are described byWolff et al. (1990) Science 247: 1465-1468 and by Sedegah et al. (1994)Immunology 91: 9866-9870. Other modes of administration include oral andtransdermal.

Vaccines of the invention can be administered as a primary prophylacticagent in, e.g., adults or in children, as a secondary prevention, aftersuccessful eradication of Gram-positive bacteria such as S. aureus in aninfected host, or as a therapeutic agent in the aim to induce an immuneresponse in a host to prevent infection by a Gram-positive bacteriumsuch as S. aureus. The vaccines of the invention are administered inamounts readily determined by persons of ordinary skill in the art. Thetreatment may consist of a single dose or a plurality of doses over aperiod of time. For example, in some embodiments, it is expected that atypical dosage for humans of a vaccine of the present invention is about1 to 25 μg of the oligosaccharide antigen, which will be bound to (anddoes not include the mass of) the protein carrier, in furtherembodiments about 1 μg to about 10 μg of the polysaccharide antigen, andin still further embodiments about 2 μg of the polysaccharide antigen.In additional embodiments, the sugar/protein ratio in the glycoconjugateor the vaccine is about 1:5 to about 1:10. Optionally, a vaccine, suchas a bioconjugate vaccine of the present invention, may include anadjuvant. Those skilled in the art will recognize that the optimal dosemay be more or less depending upon the patient's body weight, disease,the route of administration, and other factors. Those skilled in the artwill also recognize that appropriate dosage levels can be obtained basedon results with known vaccines. The number of doses will depend upon thedisease, the formulation, and efficacy data from clinical trials.

The vaccine compositions can be packaged in forms convenient fordelivery. Delivery forms compatible with entry of the immunogeniccomponent or fragment into the recipient mammal are preferred.

One embodiment of the invention is generally directed to recombinantlyproducing a vaccine for a Gram-positive organism in a Gram-negativeorganism by using a modified LPS biosynthetic pathway. This isaccomplished by inserting into a host which comprises of nucleic acidsencoding for an oligosaccharyltransferase and a protein and nucleicacids encoding for glycosyltransferases originating from at least twodifferent organisms. This embodiment is directed to geneticallyengineering an organism based on a natural organism into which areinserted nucleic acids coding for (i) a protein; (ii) anoligosaccharyltransferase, and (iii) glycosyltransferases from at leasttwo differing organisms.

In an example of such an embodiment, a glycosylated-protein product isproduced for use as a vaccine for Staphylococcus aureus. The vaccineproducts of the invention are produced in a genetically modified E. colihost. S. aureus is a Gram-positive bacterium, and has a polysaccharidecapsule. A vaccine product for this organism could be based on aglycosylated protein whose sugar section had a structure similar to thiscapsular polysaccharide.

In another aspect, the instant invention is directed to a novelbioengineering approach for producing immunogenic conjugate vaccinesthat provide advantages over classical chemical conjugation methods. Inan embodiment, the approach involves in vivo production of glycoproteinsin bacterial cells, for example, Gram-negative cells such as E. coli.

As known to a person of ordinary skill in the art, the production andpurification of glycoconjugate can vary depending on the vaccinecandidate and the combination of plasmids used. For example, whichpurification procedure to choose is known based upon the proteincarrier, the sugar component of the glycoconjugate and the intended useof the purified vaccine candidate, for example, in animals or humans.For use in humans, for example, it is known that the HIS-tag, whichwould otherwise facilitate purification, would be removed.

All publications mentioned herein are incorporated by reference in theirentirety. It is to be understood that the term “or,” as used herein,denotes alternatives that may, where appropriate, be combined; that is,the term “or” includes each listed alternative separately as well astheir combination. As used herein, unless the context clearly dictatesotherwise, references to the singular, such as the singular forms “a,”an,” and “the,” include the plural, and references to the plural includethe singular.

The invention is further defined by reference to the following examplesthat further describe the compositions and methods of the presentinvention, as well as its utility. It will be apparent to those skilledin the art that modifications, both to compositions and methods, may bepracticed which are within the scope of the invention.

EXAMPLES Example 1: Synthesis of CP5 and CP8 Polysaccharide in E. coliCells

A goal of an embodiment of the invention is to produce the CP5 and CP8antigenic polysaccharides in E. coli. As discussed above, we exploitedin an novel way, surprising in view of the prior art, the fact that theCP and O-antigen production pathways functionally overlap, a fact whichis represented in the structure of the RU (See FIGS. 1-4). The capsularglycans of CP5 and CP8 are polymers consisting of similar trisaccharideRUs of 2-Acetamido-2-deoxy-D-mannuronic acid (D-ManNAcA) and two2-Acetamido-2,6-dideoxy galactose residues with D- and L-configurations(D- and L-FucNAc). The ManNAcA residues are linked differently in thetwo serotypes; additionally, the linkage between RUs in the polymerizedglycan is different. In addition, there is an immunodominant O-acetylmodification at different positions in the two antigens (Jones, C. 2005.Revised structures for the capsular polysaccharides from Staphylococcusaureus types 5 and 8, components of novel glycoconjugate vaccines.Carbohydr Res 340:1097-106). The O11 antigen of P. aeruginosa LPS issimilar in its structure to CP5 and CP8, as the O11 antigen of P.aeruginosa LPS contains[-3)-α-L-FucNAc-(1,3)-β-D-FucNAc-(1,2)-3-D-Glc-(1-] (FIG. 4). (Knirel,Y. A., V. V. Dashunin, A. S. Shashkov, N. K. Kochetkov, B. A. Dmitrievand I. L. Hofman. 1988. Somatic antigens of Shigella: structure of theO-specific polysaccharide chain of the Shigella dysenteriae type 7lipopolysaccharide. Carbohydr Res 179: 51-60). The trisaccharide-RUsdiffer only in that the D-ManNAcA of S. aureus is replaced by a glucoseunit, there is no O-acetyl modification in P. aeruginosa O11 LPS, andthe difference in the linkage type between the 2^(nd) and 3^(rd)monosaccharide in the RU (FIG. 4).

To generate a genetic system able to synthesize the CP5 and CP8 glycanson UndPP, using the method of Dean et al., (Dean, C. R., C. V.Franklund, J. D. Retief, M. J. Coyne, Jr., K. Hatano, D. J. Evans, G. B.Pier, and J. B. Goldberg. 1999. Characterization of the serogroup O11O-antigen locus of Pseudomonas aeruginosa PA103. J Bacteriol181:4275-4284), we modified the P. aeruginosa O11 O-antigen gene clusterfrom strain PA103. The genes encoding the biosynthetic machinery forsynthesis of the stem structure consisting of UndPP-D-FucNAc-L-FuncNAcwere complemented with the S. aureus enzymes required for the completionof the S. aureus glycan (FIG. 1-4), which was also a novel use of thisprocess. Therefore, using the method of Dean et al., all the geneticelements from P. aeruginosa PA103 required for the UndPP-FucNAc-FucNAcbiosynthesis were expressed. The gene encoding the glycosyltransferaseadding the third sugar was deleted and replaced by the correspondinggenes from the cap5 or 8 clusters form S. aureus Mu50 (CP5) and MW2(CP8) with slight modifications.

The genes encoding the enzymes synthesizing the specific residues forthe S. aureus capsular polysaccharide were integrated step by step intothe O11 background according to the functions of the genes predicted bySau et al. (Sau, S., N. Bhasin, E. R. Wann, J. C. Lee, T. J. Foster, andC. Y. Lee. 1997. The S. aureus allelic genetic loci for serotype 5 and 8capsule expression contain the type-specific genes flanked by commongenes. Microbiology 143: 2395-405; O'Riordan, K. and J. C. Lee. 2004.Staphylococcus aureus capsular polysaccharides. Clin Microbiol Rev17(1): 218-34). Such steps are explained below.

The cap5I/cap8H gene product was predicted to be the glycosyltransferasethat adds the ManNAcA to UndPP-D-FucNAc-L-FuncNAc of the RU forming alinkage specific for each serotype (Sau, S., N. Bhasin, E. R. Wann, J.C. Lee, T. J. Foster, and C. Y. Lee. 1997. The Staphylococcus aureusallelic genetic loci for serotype 5 and 8 capsule expression contain thetype-specific genes flanked by common genes. Microbiology 143:2395-405.). To prove this, the activity of Cap5I and Cap8H was analyzedin E. coli in presence of a plasmid conferring production of the P.aeruginosa O11 O-antigen. Cells expressing the O11 cluster synthesizethe O11 O-antigen first on UndPP, from where it is transferred to lipidA core by the E. coli enzyme WaaL, the O-antigen ligase, forming O11specific lipopolysaccharide (LPS) (Goldberg, J. B., K. Hatano, G. S.Meluleni and G. B. Pier. 1992. Cloning and surface expression ofPseudomonas aeruginosa O antigen in Escherichia coli. Proc Natl Acad SciUSA 89(22): 10716-20). To synthesize this lipopolysaccharide, the O11O-antigen cluster from P. aeruginosa PA103 was cloned into pLAFR1 (SEQID NO: 1). Then the wbjA gene encoding the glucosyltransferase, theenzyme adding the third sugar to the O11 RU, was deleted by transposonmutagenesis. The mutated cluster (O11 wbjA::Tn50<dhfr-1>) was furthermodified by homologous recombination to eliminate the polymeraseactivity of the wzy gene, forming O11 wbjA::Tn50<dhfr-1>wzy::cat, whichdenotes the mutated SEQ ID NO: 1, in which the genes for theglycosyltransferase wbjA and the wzy polymerase of the O11 gene clusterwere inactivated. This modified cluster was expressed in W3110 ΔwecAcells, extracts were treated with proteinase K and analyzed by SDS PAGEand silver staining, according to the method disclosed in Tasi, et al.(Tsai, C. M., and C. E. Frasch. 1982. A sensitive silver stain fordetecting lipopolysaccharides in polyacrylamide gels. Anal Biochem119:115-9). The results are provided in FIG. 5A, showing silver stainingof W3110 ΔwecA extracts expressing the mutated O11 cluster from pLAFR1as described herein. The second line indicates the genes expressed fromthe inducible plasmid pEXT22. Asterisks indicate synthesized and codonoptimized genes. Different relevant glycoforms are indicated witharrows.)

Analysis resulted in two major bands in the gels (FIG. 5A, lane 1). Thesignals correspond to the unmodified lipid A core (FIG. 5A, lower band)and LPS consisting of lipid A core and two FucNAc residues as expectedin a truncated O11 RU. Upon expression of a wbjA wildtype copy from aseparate, IPTG inducible plasmid, the upper band shifted to a slowerelectrophoretic mobility, indicating the addition of a glucose residueto the truncated O11 LPS (FIG. 5A, lane 2). When the predicted S. aureusglycosyltransferases Cap5I (lane 4) and Cap8H (FIG. 5A, lane 3) wereexpressed in trans instead of WbjA, a similar shift of the glycosylatedlipid A core signal was observed, indicative of addition of amonosaccharide possibly even larger than glucose, most probably beingManNAcA. This data proves that S. aureus glycosyltransferases canelongate UndPP-D-FucNAc-L-FuncNAc glycolipid that has been synthesizedby activity of P. aeruginosa enzymes.

In this way it was also confirmed that a prerequisite for S. aureus RUassembly in E. coli is the provision of UDP-ManNAcA, because thebiosynthetic machinery is present in the S. aureus CP5/8 clusters butnot in the O11 O-antigen cluster of P. aeruginosa. All other requirednucleotide activated sugars are either provided by housekeepingfunctions of E. coli and the O11 O-antigen cluster of P. aeruginosa. E.coli is known to produce UDP-ManNAcA, the substrate for the ManNAcAglycosyltransferase, by expression of wecB and wecC. Those genes areconstitutively expressed in the cluster responsible for enterobacterialcommon antigen (ECA) biosynthesis (Meier-Dieter, U., R. Starman, K.Barr, H. Mayer, and P. D. Rick. 1990. Biosynthesis of enterobacterialcommon antigen in Escherichia coli. J Biol Chem 265:13490-13497). Thefunctional homolog for UDP-ManMAcA biosynthesis found in the CP clusterof S. aureus were found to complement the activities of wecBC asreported earlier (Kiser, K. B., N. Bhasin, L. Deng and J. C. Lee. 1999.Staphylococcus aureus cap5P encodes a UDP-N-acetylglucosamine2-epimerase with functional redundancy. J. Bacteriol 181(16): 4818-24).This shows that the production of the CP antigens in E. coli relies onthe functional expression of the wecBC genes in the host strain. Thus,to provide UDP-ManNAcA as substrate for CapSI and Cap8H in a recombinantsystem, it was confirmed that WecB and WecC have to be expressed. Insuch a system, any prokaryotic strain expressing the enterobacterialcommon antigen like E. coli wildtype strain can be used, e.g. W3110based cell types with or without a wecA deletion and with or withoutadditional wzzE deletion.

Further elongation of the S. aureus capsular polysaccharide is thoughtto be required for maximal immunological activity of the glycan. Thecap5J/cap8I genes encode the wzy homologs polymerizing the repeatingunits, and cap5K/cap8K encodes the flippase translocating theUndPP-bound trisaccharide from the cytoplasmic to the periplasmic sideof the membrane. Cap5H/cap8J encodes the O-acetyltransferase modifyingthe L-FucNAc at position 3′ or the ManNAcA at position 4′ of the RU(Bhasin, N., A. Albus, et al. (1998). “Identification of a geneessential for O-acetylation of the Staphylococcus aureus type 5 capsularpolysaccharide.” Mol Microbiol 27(1): 9-21. The acetylation is animportant determinant discriminating the immunological reactivity of thepolysaccharide (Fattom, A. I., J. Sarwar, L. Basham, S. Ennifar, and R.Naso. 1998. Antigenic determinants of S. aureus type 5 and type 8capsular polysaccharide vaccines. Infect Immun 66:4588-92). To show thatthe RUs could be elongated and acetylated, the S. aureus enzymesresponsible for polymerization and O-acetylation were expressed fromseparate plasmids in presence of the mutated O11 cluster. Extracts fromW3110 ΔwecA cells expressing the O11 wbjA::Tn50<dhfr-1 >wzy::cat clusterand different genes of the CP5 cluster were treated with proteinase Kand analyzed by SDS PAGE, electrotransfer followed by immunoblottingusing an anti CP5 sugar (obtained from J. C. Lee at the Department ofMedicine, Brigham and Women's Hospital, Harvard Medical School, Boston,Mass., USA). FIG. 5B shows the results of immunodetection of proteinaseK treated E. coli extracts separated by SDS PAGE and electrotransferusing the anti CP5 antiserum. All extracts analyzed contained a P.aeruginosa O11 cluster with deletions of the wbjA and partially(indicated by an asterisk) the wzy genes expressed from the pLAFRplasmid as described herein, and two more plasmids (pEXT22, pACT3)expressing different Cap5 proteins (as indicated) that enable CP5polymerization and O acetylation in these cells. Experimental detailssuch as inducer concentrations and expression culture incubationtemperatures are indicated.

In FIG. 5B, the results show ladder like signals typical for anO-antigen polymer in a higher molecular weight range. The differentbands represent different numbers of linearly polymerized RUs on LPS oron UndPP, both of which are stable towards proteinase K digestion.Different intensities of the ladder like structure in presence orabsence of the O-acetyltransferase were observed. Whereas strong signalswere detected in the presence of cap5H (FIG. 5B, lanes 1-4), they werevirtually absent in lanes without cap5H (FIG. 5B, lanes 5, 6). Thismeans that O-acetylation either increases recognition by the specificantiserum, or that it enhances polymerization activity by eitheraccelerating flipping or making polymerization as such more efficient orby inducing more RU production. The cap5H gene is functional whenexpressed from different backbone plasmids (FIG. 5B, lanes 1, 2 and 3,4), although signal intensity is stronger when cap5H is expressed alonefrom a separate plasmid (compare FIG. 5B lane 1 to lane 3 and FIG. 5B,lane 2 to lane 4). It is surprising and remarkable that the less IPTGwas used for induction of the S. aureus genes, the stronger the signals(compare FIG. 5B, lane 1 to land 2 and FIG. 5B, lane 3 to lane 4).

Example 2: Synthesis of CP5 and CP8 Polymer on Lipid in E. coli Cells

As high expression of the cap5 specific genes lead to lower polymerformation, an alternative expression system for the recombinant glycanswas constructed to address this problem. In detail, in a novel approachunexpected in light of the prior art, the P. aeruginosaglucosyltransferase (wbjA) and the polymerase (wzy) of O11 were replacedby the genes encoding the CP5/8-specific elements from the capsular genecluster of S. aureus Mu50/MW2 (cap5/8HIJK and parts thereof) producing asingle, chimeric gene cluster composed of P. aeruginosa O11 and S.aureus CP5 or CP8 genes (FIG. 6). The construct contained the specificgenes of S. aureus. Each was tagged for expression detection and eachcontained an introduced ribosomal binding site, and was followed by achloramphenicol resistance cassette (cat) for selection of recombinedclones resulting in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4,according to the method of Datsenko, et al. (Datsenko, K. A., and B. L.Wanner. 2000. One-step inactivation of chromosomal genes in Escherichiacoli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640-5).

FIG. 6 depicts an embodiment of a strategy of the present invention forconstruction of chimeric O11/CP5 and O11/CP8 gene clusters of thepresent invention. The S. aureus CP5 and CP8 CP clusters (top) and theP. aeruginosa PA103 rfb cluster (O11, middle) are represented aspublished (Dean, C. R., C. V. Franklund, J. D. Retief, M. J. Coyne, Jr.,K. Hatano, D. J. Evans, G. B. Pier, and J. B. Goldberg. 1999.Characterization of the serogroup O11 O-antigen locus of Pseudomonasaeruginosa PA103. J Bacteriol 181:4275-84; Sau, S., N. Bhasin, E. R.Wann, J. C. Lee, T. J. Foster and C. Y Lee. 1997. The S. aureus allelicgenetic loci for serotype 5 and 8 capsule expression contain thetype-specific genes flanked by common genes. Microbiology 143 (Pt 7):2395-405). The homologous functions of the genes are described below.Complete forward diagonals indicate the genes responsible for synthesisof the D-FucNAc-L-FucNAc disaccharide on UndPP in the two organisms;dots indicate the glycosyltransferase genes adding the thirdmonosaccharide to the RU. Wzx-like flippase genes are indicated bybroken forward diagonals, the wzy-like RU polymerase genes are indicatedby broken back diagonals. The CP5 cluster does not contain a Wzz lengthregulator (empty arrow), but a set of three genes composing the exportmachinery for capsular polysaccharide which includes the lengthregulator function in S. aureus (empty arrows). The O acetyl transferasegene, indicated by complete forward diagonals, is unique to the CPcluster. The genes required for UDP-ManNAcA biosynthesis in S. aureusare indicated in black. They are not required for production of the P.aeruginosa O-antigen. The genes responsible for the structuraldifferences of the O11, CP5 and CP8 polysaccharides are clusteredtogether in the beginning (O11: wbjA and wzy) or middle (CP5/8:cap5/8HIJK) of the respective gene clusters. The CP8 cluster is almostidentical to the CP5 cluster considering length and DNA sequence, exceptfor the middle part (cap5/8HIJK) conferring structural specificity. Thechimeric cluster was constructed by replacing wbjA and wzy genes of aplasmid borne O11 cluster with the specificity part of the CP5 (or CP8)cluster (cap5/8HIJK) and a chloramphenicol acetyltransferase cassetterepresented by the empty arrow labeled cat (cat, for selection) byhomologous recombination and classical clonings, resulting in SEQ ID NO:2, SEQ ID NO: 3, and SEQ ID NO: 4. Asterisks at the broken arrowsindicate incomplete gene sequences used for homologous recombination.The resulting two chimeric clusters are shown in the bottom panel,representing the DNA of SEQ ID NO: 3 and SEQ ID NO: 4.

To prove that the chimeric CP5 and CP8 of the present inventionsurprisingly assembles the correct RU on UndPP and assures that therepeating units are polymerized, proteinase K digestion of E. coli cells(W3310 ΔwecA) containing the full length chimeric clusters wereseparated by SDS-PAGE. Specifically, cells with a plasmid eithercontaining or lacking the chimeric CP5 gene cluster (FIG. 7A) or thechimeric CP8 gene cluster (FIG. 7B) on the pLAFR plasmid were treatedwith Proteinase K, separated by SDS-PAGE and lipids were visualized byeither silver staining (left panel in FIGS. 7A and 7B) orimmunodetection with anti CP5 or CP8 antiserum after electrotransfer tonitrocellulose membranes (right panel in FIGS. 7A and B)). Constructslacking (SEQ ID NO: 2) and containing (SEQ ID NO: 3) the flippase genecap5K were tested. The former was found to be less active in CP5 LPSproduction.

After electrotransfer and immunodetection with anti CP5 specific serum,extracts expressing the entire chimeric CP5 clusters show a ladder likesignal similar to endogenous O-antigen structures from E. coli probedwith their autologous serum (FIG. 7A, last two lanes on the right). Thisstrongly suggests that the CP5 repeating units are polymerized, thatthere is a preferred polymer length, and that the CP5 antigen istransferred to lipid A core in these cells. The same extracts werevisualized by silver staining after SDS PAGE (FIG. 7A, on the left sideof the figure, the two lanes on the right labeled as: chimeric CP5 (w/ocap5K) and chimeric CP5 showing that indeed LPS is formed consisting ofthe lipid A core of E. coli decorated with the CP5 O-antigen-likestructure. Intensity differences were obtained from extracts originatingfrom cells that expressed the CP5 chimeric cluster with or without thecap5K flippase gene. Comparison of the two extracts shows that Cap5Kexpression considerably increases the polymer production (compare middleand right lanes in both panel of FIG. 7A).

As shown in FIG. 7B, the same results were observed with a CP8 chimericcluster. Cells containing a plasmid either containing or lacking thechimeric CP8 gene cluster on the pLAFR plasmid were treated withProteinase K, separated by SDS PAGE and lipids were either detected bysilver staining (left panels) or immunodetection with anti CP8 antiserumafter electrotransfer to nitrocellulose membranes (right panel). CP8chimeric construct containing the flippase gene cap8K corresponds to SEQID NO: 4.

A further novel and surprising extension of the invention was developedby changing the plasmid backbones used for maintenance and expression ofthe chimeric cluster in E. coli. The resistance cassette in pLAFR1containing the chimeric CP5 cluster was changed from Tet to Kan.Additionally the entire CP5 chimeric cluster containing the cap5K wassubcloned into plasmid pDOC-C, according to the method of Lee et al.(Lee, D. J., L. E. Bingle, K. Heurlier, M. J. Pallen, C. W. Penn, S. J.Busby and J. L. Hobman. 2009. Gene doctoring: a method forrecombineering in laboratory and pathogenic Escherichia coli strains.BMC Microbiol 9: 252) and pACYC 177 (GeneBank accession #X06402).

As shown in FIGS. 8A and 8B, all of these plasmids conferred CP5 polymerproduction as analyzed by SDS PAGE, electrotransfer and immunodetectionwith anti CP5 specific antiserum. In FIG. 8A, total cell extracts fromcells containing different chimeric clusters were treated withProteinase K and analyzed by SDS PAGE and silver staining. The plasmidscontain different S. aureus specific genes and different resistancegenes used for antibiotic selection are indicated: Tetracycline (Tet)and HIJ, SEQ ID NO: 2; Tet HIJK, SEQ ID NO: 3, Tet and no genes, emptyplasmid control, numbers correspond to molecular weight markers. Laneslabeled Kanamycin (Kan) contains a variation of SEQ ID NO: 3 in whichthe tetracycline resistance cassette is replaced by a kanamycinresistance gene.

In FIG. 8B, the host strain was E. coli W3110 ΔwecA, as in FIG. 8A. Theleft lane in FIG. 8B corresponds to the molecular weight marker as inFIG. 8A. In FIG. 8B, total cell extracts from cells containing differentchimeric clusters were treated with Proteinase K and analyzed by SDSPAGE and silver staining (left panel) and by anti CP5 immunoblottingafter electrotransfer (right panel). The plasmids used contain thechimeric CP5 cluster indicated in SEQ ID NO: 3 either present in amodified pLAFR1 plasmid backbone containing a Kanamycin cassette insteadof tetracycline (see FIG. 8A) or in pACYC containing a chloramphenicolresistance cassette.

In addition different promoters were tested to express the chimericO11-CP5 LPS. In these tests, the host strain was E. coli W3110 ΔwecAcarrying the chimeric CP5 cluster. In this strain, the chimeric clusterreplaced wecAwzzE genes. Total cell extracts from cells containingdifferent chimeric clusters expressed from pLAFR1 were treated withProteinase K and analyzed by SDS PAGE and anti CP5 immunoblotting afterelectrotransfer. The plasmids contained O11 clusters where wbjA and wzywere replaced by different S. aureus specificity genes (with a catcassette) as indicated below the lanes in FIG. 9. In addition, the DNAin front of the cap5 specificity genes was changed and the effect onlipid glycosylation was analyzed. The effect of these different promoterregions was analyzed as depicted in FIG. 9. Wzz/wzx denotes the originalgenes (see FIG. 6) in front of the cap genes after the initialhomologous recombination (FIG. 9 corresponding to the first two lanes).These two genes were removed (FIG. 9 corresponding to the three lanes inthe middle) and replaced with the 0.6 kb region (PO121) (FIG. 9corresponding to the three last lanes) in front of the E. coli 0121O-antigen cluster encoding a strong promoter sequence. Lanes denotedwzz/wzx and HIJ in FIG. 9 were derived from cells expressing SEQ ID NO:2, lanes denoted wzz/wzx and HIJK derive from SEQ ID NO: 3. In FIG. 9,the molecular weight markers are indicated on the left of the gel frame.

As indicated FIG. 9, the results showed that a relevant promoteractivity resides in the wzx gene (FIG. 9 first two lanes—wzz/wzx) andthat it can be functionally replaced by a constitutive promoter from E.coli, e.g. the serovar O121 wb promoter (PO121 last three lanes in FIG.9), without losing LPS production. Taken together, these results meanthat the O11 and S. aureus elements for O11 O-antigen and CP5 capsularpolymer production as described herein can be combined in many differentE. coli expression systems resulting in production of recombinant S.aureus polysaccharide.

These results showed for the first time the production in E. coli of acapsular polysaccharide structure originating from a Gram-positiveorganism. This means that it was possible, contrary to prior art andconventional expectations, to combine the enzymes of the O11 cluster andthe enzymes of S. aureus cap cluster to build up a chimericpolysaccharide, i.e. that the enzyme work together on the same structurein vivo.

Example 3: Molecular Structure Confirmation of the Recombinant Glycans

To confirm the activity of the chimeric CP5/O11 cluster in E. coli on amolecular level, a novel method allowing the analysis of UndPP linkedsugars by using fluorescent labeling of the sugar at reducing end with2-Aminobenzamide (2-AB) was developed. To enhance the analysisresolution, chimeric clusters were used containing deletions thatincreased the amount of unpolymerized RUs. Glycolipids from different E.coli cells expressing the chimeric cluster contained in the pLAFR1plasmid and lacking the cap5K flippase (SEQ ID NO: 2) were analyzed asdescribed below.

To extract UndPP-linked glycans, E. coli cells were washed with 0.9%NaCl and lyophilized. The dried cells were extracted once with 30 mlorganic solvent (85 to 95% Methanol=M). The lyophilized cell pellet wasfurther extracted twice with 5 ml Chloroform:Methanol:Water(C:M:W=10:10:3; v/v/v). The (M) extract was converted with chloroformand water to a final ratio of 3:48:47 (C:M:W). The 10:10:3 (C:M:W)extract was converted to a two-phase Bligh/Dyer (Bligh, E. G. and W. J.Dyer. 1959. A rapid method of total lipid extraction and purification.Can J Biochem Physiol 37(8): 911-7) system by addition of water,resulting in a final ratio of 10:10:9 (C:M:W). Phases were separated bycentrifugation and the upper aqueous phase was kept for furtherprocessing.

To purify the extracted glycolipids, aqueous phase was subjected to atC₁₈ Sep-PAK cartridge. The cartridge was conditioned with 10 mlmethanol, followed by equilibration with 10 ml 3:48:47 (C:M:W). Afterloading of the sample, the cartridge was washed with 10 ml 3:48:47(C:M:W) and eluted with 5 ml methanol and 5 ml 10:10:3 (C:M:W). Thecombined elutions were dried under N₂. The glycolipid samples werehydrolyzed by dissolving the dried samples in 2 ml n-propanol:2 Mtrifluoroacetic acid (1:1), heating to 50° C. for 15 min, and thenevaporating to dryness under N₂ (Glover, K. J., E. Weerapana and B.Imperiali. 2005. In vitro assembly of the UndPP-linked heptasaccharidefor prokaryotic N-linked glycosylation. Proc Natl Acad Sci USA 102(40):14255-9). The dried samples were labeled with 2-AB and the glycancleanup was performed using the paper disk method as described (Bigge,J. C., T. P. Patel, J. A. Bruce, P. N. Goulding, S. M. Charles, R. B.Parekh. 1995. Nonselective and efficient fluorescent labeling of glycansusing 2-amino benzamide and anthranilic acid. Anal Biochem 230(2):229-38; Merry, A. H., D. C. Neville, L. Royle, B. Matthews, D. J.Harvey, R. A. Dwek and P. M. Rudd. 2002. Recovery of intact2-aminobenzamide-labeled O-glycans released from glycoproteins byhydrazinolysis. Anal Biochem 304(1): 91-9). The 2-AB labeled glycanswere separated by HPLC using a GlycoSep-N normal phase column accordingto Royle et al. but modified to a three solvent system (Royle, L., T. S.Mattu, E. Hart, J. I. Langridge, A. H. Merry, N. Murphy, D. J. Harvey,R. A. Dwek, P. M. Rudd. 2002. An analytical and structural databaseprovides a strategy for sequencing O-glycans from microgram quantitiesof glycoproteins. Anal Biochem 304(1): 70-90). Solvent A was 10 mMammonium formate pH 4.4 in 80% acetonitrile. Solvent B was 30 mMammonium formate pH 4.4 in 40% acetonitrile. Solvent C was 0.5% formicacid. The column temperature was 30° C. and 2-AB labeled glycans weredetected by fluorescence (excitation λex=330 nm, emission λem=420 nm).Gradient conditions were a linear gradient of 100% A to 100% B over 160min at a flow rate of 0.4 ml/min, followed by 2 min 100% B to 100% C,increasing the flow rate to 1 ml/min. The column was washed for 5 minwith 100% C, returning to 100% A over 2 min and running for 15 min at100% A at a flow rate of 1 ml/min, then returning the flow rate to 0.4ml/min for 5 min. Samples were injected in water.

Dried fractions were resuspended in 5 ul 10% acetonitrile (ACN), 0.1%trifluoro acetic acid (TFA) and mixed 1:1 with matrix solution (40 mg/mlDHB in 50% ACN, 0.1% TFA) on the target plate. MS and MS/MS data weremanually acquired in the positive ion mode on an Ultraflex-IIMALDI-ToF/ToF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany).MS/MS were obtained using the LIFT method. A standard peptide mixture(Bruker Daltonik GmbH) was used for external calibration. Spectra wereexported using the Flex Analysis software (Bruker Daltonik GmbH) andmanually analyzed.

Methanol extracts from E. coli W3110 ΔwecA (CP5) containing plasmidswith (thick line) or without (thin, dashed line) the chimeric clusterswere purified over tC18 cartridges and analyzed by normal phase HPLC.The fractions corresponding to the peaks shown in FIG. 10A found at 37′,40′ and 45′ elution were analyzed by MALDI-MS/MS. Samples eluting at 37and 40 minutes were identified as recombinant CP5 RUs with and withoutthe O-acetyl group attached, respectively. Sample eluting at 45 minuteswas identified as non-acetylated S. aureus RU structure elongated by onedeoxy-N-acetylhexosamine (as shown in FIG. 11E). In the CP5 chimericcluster, cap5HIJ replaced the wbjA and wzy genes of the O11 cluster onpLAFR. The replacement contained the cat cassette in addition to thecap5HIJ genes (SEQ ID NO: 2).

Methanol extracts from E. coli W3110 ΔwecAwzzE containing plasmids with(thick line) or without (thin, dashed line) the chimeric cluster werepurified over tC18 cartridges and analyzed by normal phase HPLC. FIG.10B shows the results of HPLC analysis of recombinant RU of CP8 producedusing a chimeric cluster (SEQ ID NO: 4 without polymerase). Peaksspecific for cells expressing the recombinant sugar were identified at23′, 32′, 38′ and 45 of elution, collected and analyzed by MALDI-MS andMALDI-MS/MS. In the CP8 chimeric cluster, cap8HJK replaced the wbjA andwzy genes of the O11 cluster, i.e. a construct without the polymerase toaccumulate single RU for analysis. The replacement contained the catcassette in addition to the cap genes.

FIG. 11A shows the results of MALDI-MS/MS analysis of the specific peakgenerated by expression of an embodiment of the chimeric CP5 cluster ofthe present invention in E. coli eluting at 37 minutes. The major massm/z=772 ([M+H]⁺) was selected and analyzed by MS/MS, which shows afragmentation pattern consistent with the acetylated CP5 RU structurethat was expected in light of the invention disclosed in thisspecification. The O-acetylated species are characterized by a specificloss of 42 plus the mass of the monosaccharide FucNAc (dHexNAc(OAc)) atthe middle position of the RU. Fragment ions are indicated according tothe nomenclature of the consortium for functional glycomics, CFG(www.functionalglycomics.org/static/consortium/Nomenclature.shtml).2-AB, 2-aminobenzamide. The legend for the fragment ions is given in theinset of FIG. 11A.

FIG. 11B shows the results of MALDI-MS/MS analysis of the specific peakgenerated by expression of an embodiment of the chimeric CP5 cluster ofthe present invention in E. coli eluting at 40 minutes. The major massof m/z=730 ([M+H]⁺) was selected and analyzed by MS/MS, which showsfragmentation ion series consistent with the non-acetylated CP5 RUstructure that was expected in light of the invention disclosed in thisspecification. 2-AB, 2-aminobenzamide. The legend for the fragment ionsis given in the inset of FIG. 11B.

FIG. 11C shows the results of MALDI-MS/MS analysis of the specific peakgenerated by expression of an embodiment of the chimeric CP8 cluster ofthe present invention in E. coli eluting at 32 minutes. A major mass ofm/z=794 ([M+Na]⁺) was selected and analyzed by MS/MS, which showsfragmentation ion series consistent with the acetylated CP8 RU structurethat was expected in light of the invention disclosed by thisspecification. The O-acetylated species are characterized by a specificloss of 42 plus the mass of the monosaccharide ManNAcA (HexNAcA(OAc)) atthe outermost position of the RU. Fragment ions are indicated accordingto the nomenclature of the CFG. 2-AB, 2-aminobenzamide. The legend forthe fragment ions is given in the inset of FIG. 11C.

FIG. 11D shows the results of MALDI-MS/MS analysis of the specific peakgenerated by expression of an embodiment of the chimeric CP8 cluster ofthe present invention in E. coli eluting at 38 minutes. The mass ofm/z=730 ([M+H]⁺) was selected and analyzed by MS/MS, which showsfragmentation ion series consistent with the non-acetylated CP8 RUstructure that was also expected in light of the invention disclosed inthis specification. Additional analysis showed that the later elutingpeaks (shown in FIG. 10A at 40 min and FIG. 10B at 38 min) contain thenon-O-acetylated trisaccharides of CP5 and 8 RUs. Fragment ions areindicated according to the nomenclature of the CFG. 2-AB,2-aminobenzamide. The legend for the fragment ions is given in the insetof FIG. 11D.

MS results showed that the masses and fragmentation ion series are inagreement with the molecular structure of the CP5 RU oligosaccharidewith the O acetylation of the middle FucNAc residue (i.e., the peak at37′ in FIG. 10A and in FIG. 11A) or without the O acetylation of themiddle FucNAc residue (i.e., the peak 40′ in FIG. 10A and in 11B). Thesignal at 45 minutes in FIG. 10A was identified as a tetrasaccharide,which is further analyzed below. The same analysis was repeated with thechimeric CP8 cluster that lacked the polymerase gene. In such extracts,signals consistent with the O-acetylated RU structure expected in lightof the invention disclosed in this specification were found at 23′ and32′ of elution, as shown FIGS. 10B and 11C. The presence of twodifferent elution times for the same glycan sequence as identified byMALDI-MS/MS indicates an O-acetyl migration event taking place duringsample preparation. Non-acetylated RUs were identified for CP5 and CP8extracts at 40′ and 38′, as shown in FIGS. 11B and D, respectively. TheCP5 and CP8 RU structures were present in different E. coli strains,including for example, W3110, W3310 ΔwecA, W3110 ΔwecAwzzE, and W3110ΔwecAwzzE ΔwaaL.

Example 4: Improvement of the Repeating Unit Structure and its Analysis

The HPLC peak shown in FIG. 10B eluting at 45 minutes, derived from E.coli cells expressing the chimeric CP8 cluster (SEQ ID NO: 4) butlacking the wzy polymerase gene cap8I, was also analyzed by MALDI-MS/MS.The most intense ion in the full scan MS was m/z=939 ([M+H]⁺) andsequence analysis was performed by MS/MS. The results of this MS/MSanalysis are shown in FIG. 11E, and present a fragmentation ion seriesconsistent with the non acetylated S. aureus capsular RU extended by amass of a deoxy-N-acetylhexosamine at the non-reducing end, as expectedin light of the invention disclosed in this specification. Fragment ionscorresponding to the hypothetical structures are indicated according tothe nomenclature of the CFG above the peaks. 2-AB, 2-aminobenzamide. Thelegend for the fragment ions is given in the inset of FIG. 11E.

The result shown in FIG. 11E suggested that an E. coliglycosyltransferase was able to modify the ManNAcA residue of the CP8RU. Such an altered RU most probably would not be polymerized by cap8I.Analysis of the glycosyltransferase specificities in the E. coli hostW3110 indicated that an enzyme from the ECA cluster may interfere withthe recombinant sugar, specifically the wecF gene product, a putative4-N-acetylfucosamine transferase. WecF naturally adds a4-N-acetylfucosamine onto ManNAcA comprised in ECA, most likely theenzyme could also elongate CP8 and CP5 RU.

To solve this problem, another novel approach was developed.Specifically, genes of the ECA cluster located downstream of the wecCgene including wecF were deleted. This was accomplished using the methoddescribed by Datsenko et al. (Datsenko, K. A. and B. L. Wanner (2000).“One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products.” Proc Natl Acad Sci USA 97(12): 6640-6645).Different E. coli expression hosts were deleted in the waaL andrmlB-wecG gene regions and in some strains in wecA-wzzECA as well.Sep-PAK Purified extracts (Methanol and 10:10:3 extracts) from thesemutated cells expressing the polymerase mutant CP8 chimeric cluster wereanalyzed by normal phase HPLC as described above.

FIG. 11F presents the results of HPLC analyses of methanol extracts fromE. coli W3110 ΔwaaL cells expressing the polymerase mutant of SEQ ID NO:4 (thin, dashed line) compared to cells with an additional deletion ofthe ECA cluster genes rmlB-wecG (W3110 ΔwaaL ΔrmlB-wecG::cat) (thickline). Extracts were purified over tC18 cartridges and analyzed bynormal phase HPLC. As shown in FIG. 11F, the major peak appearing at 45′in FIG. 10B was absent resulting in specific peaks for the acetylatedand non acetylated CP8 RUs (FIG. 11F) indicating that one of the ECAglycosyltransferases—most probably wecF—is responsible for the aberrantelongation phenotype. Similar results were obtained when the CP5chimeric cluster was tested in different strains. This implies thatdeleting E. coli borne glycosyltransferases and enzymes required fornucleotide activated sugar biosynthesis is a possible strategy foroptimizing quality and quantity of recombinantly producedpolysaccharides in E. coli. Target enzymes most likely would be encodedin the O-antigen cluster, the ECA cluster, and the colanic acid orcapsule clusters.

Further evidence for the quality of the recombinant polysaccharidelinked to UndPP was obtained from an optimized normal phase HPLCanalysis of Sep-PAK purified, fluorescently labeled glycolipid extractsfrom chromosomally optimized expression hosts as described above. Foroptimal performance of the Sep-PAK columns for purification of chargedCP5 and CP8 oligo- and polysaccharide-linked lipids, tert-butyl ammoniumphosphate (TBAP) was added to the extracts before loading on the Sep-PAKcartridges. As reported by Trent, et al., the cation of this saltimproves column binding of charged compounds by shielding negativecharges with hydrophobic butyl chains (Trent, M. S., A. A. Ribeiro, etal. (2001). “Accumulation of a polyisoprene-linked amino sugar inpolymyxin-resistant Salmonella typhimurium and Escherichia coli:structural characterization and transfer to lipid A in the periplasm.” JBiol Chem 276(46): 43132-43144.). This optimized method was applied tothe CP5 and CP8 samples obtained by methanol extraction from cellsexpressing CP5 or CP8 chimeric clusters containing a polymerase.

FIG. 11G provides the results of HPLC analysis showing the full CP5glycan repertoire present on UndPP in E. coli cells. Methanol extractsfrom E. coli W3110 ΔwaaL ΔwecAwzzECA ΔrmlB-wecG::cat either expressingthe chimeric CP5 cluster SEQ 3 (solid line) or an empty plasmid control(dashed line) were solid-phase extracted on Sep-PAK cartridges andtreated with mild acid to hydrolyse sugars from UndPP. The resultingmaterial was reacted with 2AB by reductive amination to label reducingends of the glycans and analyzed by normal phase HPLC. Signals presentin the solid line but not in the dashed line represent CP5 specificmaterial. Capital letters indicate peaks containing polymers of theacetylated and/or non-acetylated CP5 RU as identified by MALDI-MS/MS ofthe collected fractions. The legend of FIG. 11G indicates the proposedmolecular structures as deduced from MS/MS analysis. It should be notedthat acetylated and non-acetylated RU polymers shown for MS/MS confirmedstructures of the same polymerization degree group together in thechromatogram as indicated by thick bars. Capital letters show thefollowing lengths: A and B: one RU; C, D and E: two RUs; F and G: threeRUs; and H: four RUs. The broad peak between 95′ and 125′ in FIG. 11Gmost probably represents 5 or more polymerized RUs not resolved by thecolumn.

FIG. 11H presents further HPLC results, showing acetylated CP5 glycansand RU homogeneity. To prepare this HPLC analysis, 2AB labeled glycansamples of E. coli W3110 ΔwaaL ΔwecAwzzECA ΔrmlB-wecG::cat expressingthe chimeric CP5 cluster SEQ ID NO.: 3 (prepared according to theprocedures described above with reference to FIG. 11G) were treated withNaOH in aqueous solution and re-labeled. As showing in FIG. 11H, samplesbefore (dashed) and after (solid line) alkali treatment were analyzed byHPLC. Numbers in FIG. 11H indicate the putative numbers of RUs in thecorresponding peaks. It should be observed that, in FIG. 11H, theacetylated peaks shown in FIG. 11G unify in the signal fromnon-acetylated polymer, and that deacetylation resolved the RU units inthe elution times after 95 minutes.

FIG. 11I provides the results of HPLC analysis showing the CP8 glycanrepertoire present on UndPP in E. coli cells. Methanol extracts from E.coli W3110 ΔwaaL ΔwecAwzzECA ΔrmlB-wecG::cat either expressing thechimeric CP8 cluster SEQ ID NO.: 4 (solid line) or an empty plasmidcontrol (dashed line) were solid-phase extracted on Sep-PAK cartridgesand treated with mild acid to hydrolyse sugars from UndPP. The resultingmaterial was reacted with 2AB by reductive amination to label reducingends of the glycans and analyzed by normal phase HPLC. Signals presentin the solid line but not in the dashed line represent CP8 specificmaterial. Putative structures of acetylated and/or non-acetylated CP8 RUas identified by MALDI-MS/MS of the collected fractions are indicated.Note that as in the HPLC results with CP5 shown in FIG. 11G, acetylatedand non acetylated CP8 RU polymers of the same polymerization degreegroup together in the chromatogram of FIG. 11H as indicated by thickbars. Material detected after 110′ represents longer CP8 polymers.

FIG. 11J presents further HPLC results, showing deacetylation of CP8glycans and RU homogeneity. 2AB labeled glycan samples from E. coliW3110 ΔwaaL ΔwecAwzzECA ΔrmlB-wecG::cat expressing the chimeric CP8cluster SEQ ID NO.: 4 were treated with NaOH in aqueous solution andre-labeled. Samples before (dashed) and after (solid line) alkalitreatment were analyzed by HPLC. Numbers indicate the putative numbersof RUs in the corresponding peaks. It should be noted that theacetylated peaks largely vanish and that signals of non-acetylatedpolymer increase, and that deacetylation resolved the RU units in theelution times after 110 minutes.

FIGS. 11H and 11J show HPLC results indicative of the characteristicladder-like banding pattern of O-antigens when alkali treatment wasperformed on these CP5 and CP8 samples to remove the acetylationmodifications from the oligo- and polysaccharides. The results showdiscrete sharp peaks with constantly decreasing elution time increments.This implies that such analyzed carbohydrate chains are linear polymerscomposed of identical RUs. This data shows that the recombinant CP5 andCP8 sugars produced in E. coli are regularly polymerized and partiallyacetylated. Non-acetylated CP5 and CP8 polymers elute similarly from theHPLC column as expected from their similarity in structure; however thenormal phase chromatography also reveals differences: for example, CP5polymerizes to a lesser extent than CP8, and acetylation is morefrequent in CP5; in the RU lengths above 4, CP5 has a clear preferencefor making polymers of 7 RUs, whereas CP8 polymerizes to a broaderdegree; and as indicated by the HPLC and MS/MS results, CP5 is moreefficient for glycan production than CP8.

In wzy dependent polymerization pathways, it has been reported byMarolda, et al., that a specific enzyme (wzz or cld for chain lengthdeterminant) is responsible for determining the average number of RUpolymerization steps performed (Marolda, C. L., L. D. Tatar, et al.(2006). “Interplay of the Wzx translocase and the correspondingpolymerase and chain length regulator proteins in the translocation andperiplasmic assembly of lipopolysaccharide o antigen.” J Bacteriol188(14): 5124-5135.). Wzz enzymes cause a specific repeat numberaverages, e.g. short, long and very long sugar polymers and are known totransfer their length specificity to exogenous polysaccharide pathways.The lengths and amounts of the CP8 glycolipids were analyzed in theproduction strain resulting in longer and lower amount of this sugar. Toincrease the amount of molecules and thereby the sugar transferefficiency for protein glycosylation, a downregulation of the CP8 sugarlength was performed using a specific Wzz enzyme.

To test the effect of a Wzz protein on the size and amounts of CP8sugars on lipid, coexpression of Wzz from E. coli wzz 07 was performedfrom a separate plasmid (SEQ ID NO: 19). FIG. 11K presents the resultsof this test. Methanol extracts from E. coli W3110 ΔwaaL ΔwecAwzzECAΔrmlB-wecG::cat either expressing the chimeric CP8 cluster SEQ ID NO: 4and a plasmid borne, IPTG inducible copy of wzzO7 (SEQ ID NO: 21, solidline), or an empty plasmid control (dashed line) were solid phaseextracted on Sep-PAK cartridges and treated with mild acid to hydrolysesugars from UndPP. 2AB labeled glycans were analyzed by normal phaseHPLC. Alkali treatment of the CP8 sample showed that more than 85% ofthe area between 95 and 115′ represents 7 or 8 RU polymers of CP8,indicating a wide variety of acetylation. These results also indicatethat the chimeric CP8 cluster induced: a) an intensification in repeatnumbers of the most abundant glycan from 7 to 8, and b) a higher overallintensity of fluorescent signal as judged from the area under thechromatogram.

Alkali treatment confirmed the acetylation of the shortened glycan as inFIGS. 11I and 11J indicating that a recombinant polysaccharide's lengthcan be regulated by a foreign Wzz enzyme. It is also possible toregulate the capsular sugar polymer length by an O-antigen derived Wzzenzyme. Furthermore, different promoters in front of the chimericcluster when present on a plasmid cause different expression levels anddifferent degrees of polymerization.

Example 5: Protein Glycosylation with the CP5 and CP8 Glycans andProduct Characterization

Different variants of the chimeric cluster were tested for bioconjugateproduction. The chimeric O11/CP5 gene clusters (SEQ ID NO: 2 and 3),which contain different variants of S. aureus specificity regions in theO11 O-antigen cluster in place of wbjA and wzy, were expressed in thehost strain E. coli W3110 ΔwaaL zwecAwzzE::cat in the presence of PglB(SEQ ID NO: 27?) and EPA (SEQ ID NO: 13). W3110 ΔwaaL ΔwecAwzzE::cathost cells expressed EPA with two glycosylation sites (from SEQ ID NO:13) and PglB (SEQ ID NO: 27) from separate plasmids in addition to thepLAFR1 plasmid with the O11 O-antigen cluster where the wbjA and wzygenes were replaced with different cap5 gene sets (and the cat cassette,SEQ ID NO: 2 and SEQ ID NO: 3).

The EPA protein is expressed containing: a) a N-terminal signal peptidesequence for export to the periplasm, b) two bacterial N-glycosylationconsensus sequences engineered into the protein sequence (SEQ ID NO: 13)as set forth in Example 10 of WO 2009/104074, incorporated by referenceherein in its entirety, and c) a hexa histag for purification. The cellswere grown in 5 L Erlenmeyer flasks in LB medium. An overnight culturewas diluted to OD_(600 nm)=0.05. At OD_(600 nm) around 0.5, PglBexpression was induced by addition of 1 mM IPTG and EPA expression wasinduced by addition of arabinose (0.2% final concentration). The cellswere grown for 4 hours, induction was repeated and cells were grown foraround additional 16 hours. Cells were pelleted by centrifugation; thecells were washed and suspended in 0.2 vol sucrose buffer, pelleted, andlysed by osmotic shock. The spheroplasts were pelleted bycentrifugation, and the periplasmic proteins were loaded on a Ni²⁺affinity chromatography. EPA-CP5 bioconjugate without and with the S.aureus flippase gene cap5K (SEQ ID NO: 2 and 3) was eluted by 0.5Mimidazole, and eluted peaks were pooled and analyzed by SDS PAGE andstained by Coomassie and silver (FIG. 12).

FIG. 12 presents the SDS PAGE results. The left panel shows thecoomassie stain, and the right panel shows the silver stain. The numbersin the middle indicate the sizes of the molecular weight marker. Theletters below the lanes indicate the genes that were present in thechimeric cluster expressed in the strains used for bioconjugateproduction. The host strain was E. coli W3110 ΔwaaL ΔwecAwzzE::cat. Theresults show protein signal at 70 kDa (electrophoretic mobility) mostlikely corresponding to unglycosylated EPA, and a ladder of bands above(100-170 kDa). The ladder likely corresponds to EPA protein glycosylatedwith the CP5 recombinant S. aureus glycan. In addition, the resultsindicate that including the flippase gene in the system increases theglycoprotein yield (middle and right lanes).

In a separate analysis, CP5-EPA bioconjugate was produced in E. coliW3110 ΔwaaL ΔwecAwzzE::cat by co-expression of the chimeric CP5 genecluster (SEQ ID NO: 3), PglB (SEQ ID NO: 27) from plasmid pEXT21 and EPA(containing two glycosylation sites, SEQ ID NO: 13) from separateplasmids. To obtain a more controlled process for bioconjugateproduction, the cells were grown in a 2-L bioreactor to anOD_(600 nm)=30 at 37° C., and expression of PglB and EPA was induced bythe addition of 1 mM IPTG and 0.2% arabinose. The cells were grown for18 h at 37° C. under oxygen-limiting conditions. The cells were pelletedby centrifugation, washed and resuspended in 25% sucrose buffer at anOD_(600 nm)=200, after 30 min. incubation at 4° C., the suspension waspelleted, and lysed by osmotic shock. The spheroplasts were pelleted bycentrifugation, and the periplasmic proteins present in the supernatantwere loaded on a Ni²⁺ affinity chromatography. Glycosylated andunglycosylated EPA were eluted from the affinity column by 0.5 Mimidazole and loaded on a SourceQ anionic exchange column. GlycosylatedEPA was separated from unglycosylated EPA by applying a gradient ofincreasing concentration of NaCl.

As shown in FIG. 13A, the purified glycosylated EPA (CP5-EPA) wasseparated by SDS PAGE and stained by Coomassie (left lane) ortransferred to nitrocellulose membranes and incubated with either antiCP5 antibodies (middle lane) or anti EPA antibodies (right lane). Thepurified bioconjugate was recognized by the EPA-specific antibodies(right lane), as well as the CP5-specific polyclonal antiserum (middlelane). The arrow indicates the position in the gel from where a piecewas cut and used for trypsinization and analysis of glycopeptides byMALDI-MS/MS. FIG. 13B presents the MALDI-MS/MS of M/Z masses found forthe glycosylation site in trypsinized peptide DNNNSTPTVISHRN-glycosidically linked to the O-acetylated RU mass (m/z=2088 ([M+H]⁺)).MS/MS analysis of the m/z=2088 shows partial fragmentation of the sugarmoiety as indicated. The inset illustrates the RU structure attached tothe peptide derived from trypsinization of purified CP5-EPA from FIG.13A. Sequential losses of ManNAcA (HexNAcA, 217 Da) and acetylatedFucNAc (dHexNAc(OAc), 229 Da) support the expected glycan structure.FIG. 13C presents the MALDI-MS/MS of M/Z masses found for theglycosylation site in trypsinized peptide DQNR N-glycosidically linkedto the O-acetylated RU mass (m/z=1165 ([M+H]⁺)). MS/MS analysis ofm/z=1165 shows the full Y-ion fragmentation ion series consistent withthe CP5 RU structure. The inset illustrates the RU structure attached tothe peptide derived from trypsinization of purified CP5-EPA from FIG.13A. Sequential losses of ManNAcA (HexNAcA, 217 Da), acetylated FucNAc(dHexNAc(OAc), 229 Da), and FucNAc (dHexNAc, 187 Da) are shown,confirming the expected glycan structure on the peptide DQNR (m/z=532 Da([M+H+])).

In FIG. 13D the CP8 bioconjugate in E. coli was produced using the samestrategy as production of the CP5 bioconjugate. CP8-EPA bioconjugate wasproduced in E. coli by co-expression of the chimeric CP8 gene cluster(SEQ ID NO: 4), PglB (within the pEXT21 plasmid (SEQ ID NO: 27)), andEPA containing two glycosylation sites (SEQ ID NO: 13). Cells were grownin a bioreactor with a starting volume of 7 L in semi-defined mediumcontaining glycerol, peptone and yeast extract as C-sources. Cells weregrown at 37° C. in batch or pulsed-batch mode to an OD_(600 nm) of 30,and expression of PglB and EPA was induced by the addition of 1 mM IPTGand 10% arabinose. After induction, cells were further cultivated infed-batch mode for a period 15 hours under oxygen-limiting conditions.Cells were pelleted by centrifugation; the cells were washed andsuspended in 0.2 vol sucrose buffer, pelleted, and lysed by osmoticshock. The spheroplasts were pelleted by centrifugation, and theperiplasmic proteins were loaded on a Ni²⁺affinity chromatography.Glycosylated and unglycosylated EPA were eluted from the affinity columnby 0.5 M imidazole and loaded on a SourceQ anionic exchange column.Glycosylated EPA was separated from unglycosylated EPA by applying agradient of increasing concentration of NaCl.

As depicted in FIG. 13D, the purified protein was separated by SDS PAGEand stained by Coomassie (left lane) or transferred to nitrocellulosemembranes and incubated with either anti CP8 antibodies (right lane) oranti EPA antibodies (middle lane).

Different strategies for further improving the glycosylation system weretested. In one strategy, to reduce the plasmid number in the productionsystem to lower the burden of an additional antibiotic as well asmaintaining an extra plasmid, the expression cassette for pglB wascloned into the plasmid containing the chimeric clusters for CP5 (SEQ IDNO: 17) and CP8 (SEQ ID NO: 18). The expression cassette is composed ofthe intergene region present between galF and wbqA of the E. coli 0121genome (for a promoter sequence), and the pglB sequence downstream ofthis. This expression cassette was cloned immediately downstream of theCP5 and CP8 chimeric clusters. We tested E. coli W3110 ΔwaaLΔwecAwzzECA::cat containing the chimeric CP5 cluster (SEQ ID NO: 3) andpglB (SEQ ID NO: 27) on either separate plasmids or on the same plasmid(SEQ ID NO: 17). In addition, EPA (SEQ ID NO: 13) was expressed from aplasmid under the control of an arabinose inducible promoter. The cellswere grown in 5 L Erlenmeyer flasks in LB medium at 37° C. An overnightculture was diluted to OD_(600 nm)=0.05. At OD₆₀₀ nm around 0.5 PglBexpression was induced by addition of 1 mM IPTG and EPA expression wasinduced by addition of arabinose (0.2% final concentration). The cellswere grown for 4 hours, induction was repeated and cells were grown foraround an additional 16 hours. The culture was pelleted bycentrifugation; the cells were washed and suspended in 0.2 vol sucrosebuffer, pelleted, and lysed by osmotic shock. The spheroplasts werepelleted by centrifugation, and the periplasmic proteins were loaded ona Ni²⁺ affinity chromatography. EPA-CP5 was eluted by 0.5M imidazole,and eluted peaks were pooled and analyzed by SDS PAGE and by Coomassie.FIG. 13E depicts the SDS PAGE results. Cells containing either 3 (left)or 2 plasmids (right lane) for glycoconjugate production are shown. Theresults show that glycolipid and conjugate production for CP5-EPA wasmaintained.

A further optimization of the system was the integration of the wzz(polymer length regulator) protein sequence in the plasmids used forprotein glycosylation. Exemplified by the system producing CP8-EPA, wzzwas integrated into the plasmid borne chimeric CP8 cluster (SEQ ID NO:19) and downstream of the epa gene within the expression plasmid for thecarrier protein (SEQ ID NO: 20). CP8-EPA bioconjugate was produced in E.coli W3110 ΔwaaL ΔwecAwzzECA ΔrmlB-wecG::cat comprising 2 plasmids: oneplasmid contained in addition to the chimeric CP8 gene cluster a copy ofthe wzz 07 gene and a DNA cassette for the constitutive expression ofthe pglB gene (SEQ ID NO: 19); the second plasmid contained first thegene for expression and secretion of the detoxified EPA proteincontaining two glycosylation sites, and second a wzzO7 copy under thecontrol of the same promoter (SEQ ID NO: 20). The resulting strain, E.coli W3110 ΔwaaL ΔwecAwzzECA ΔrmlB-wecG::cat, containing the mentionedplasmids was grown in a bioreactor with a starting volume of 7 L insemi-defined medium containing glycerol, peptone and yeast extract asC-sources. Cells were grown in batch or pulsed-batch mode to anOD_(600 nm) of 30, and expression of PglB and EPA was induced. Afterinduction, cells were further cultivated in fed-batch mode for a period15 hours under oxygen-limiting conditions and collected bycentrifugation. Cells were pelleted by centrifugation; the cells werewashed and suspended in 0.2 vol sucrose buffer, pelleted, and lysed byosmotic shock. The spheroplasts were pelleted by centrifugation, and theperiplasmic proteins were loaded on a Ni²⁺ affinity chromatography.Glycosylated and unglycosylated EPA were eluted from the affinity columnby 0.5 M imidazole. Formation of glycoconjugate CP8-EPA is shown in FIG.13F by Coomassie and western blot using anti his and anti CP8 antisera.FIG. 13F shows the results of SDS PAGE separation of the purifiedprotein and analysis by Coomassie staining (left lane) or transferred tonitrocellulose membranes and probed with either anti histag antibodies(middle lane) or anti CP8 antibodies (right lane).

Characterization of the CP5-EPA glycoconjugate was further refined byvarious analytical methods. CovalX (Schlieren, Switzerland) performedHigh Mass MALDI analysis of a purified CP5-EPA sample produced using the3 plasmid system as used in the analyses depicted in FIG. 13A in W3110ΔwaaL ΔwecAwzzECA::cat. FIG. 14A depicts the High Mass MALDI results. A⁺and B⁺ point towards mass protein species ([M+H]⁺) corresponding tounglycosylated EPA and glycosylated EPA, respectively. Oligomeric formsmay be present at higher molecular weight and signals in the low MW areaare contaminants or degradation products. The results presented in FIG.14A show that the above protein preparation contained a largelymonomeric protein population which is 4 kDa larger than the EPA proteinalone, indicative of a medium sugar length of 5.2 repeating units. Thisis in agreement with the sugar length of 5-7 of the major glycoconjugateform in the preparation as analyzed by SDS-PAGE, Coomassie brilliantblue staining and counting the repeating units in the major conjugateform (see FIGS. 7, 8, and 13A).

CP5-EPA was further characterized by size exclusion chromatography(SEC-HPLC). We used the 3 plasmid system in W3110 ΔwaaL ΔwecAwzzECA::catas used in the analyses depicted in FIG. 13A. The sample was purified byanionic exchange chromatography to remove unglycosylated EPA. Analysiswas performed on a Supelco TSK G2000SWXL column. FIG. 14B shows theresults of the SEC-HPLC analysis of the purified CP5-EPA sample. The UVtrace measured at 280 nm is shown. The thick solid line derives fromanalyzing 3.25 g purified CP5-EPA, the thin line was obtained from 5 gpurified, unglycosylated EPA. A major, homogenous peak at 11.5 minutesof elution is shown, whereas unglycosylated EPA eluted at 12.9 minutes(FIG. 14B). Calculation of the hydrodynamic radii of the two moleculesresulted in a size of 42 kDa for unglycosylated EPA and 166 kDa forglycosylated EPA. This indicates that glycosylated EPA appears as anelongated, monomeric protein in solution as expected due to the linearstructure of the glycan.

Our analyses therefore confirmed that the CP5-EPA bioconjugate consistsof the EPA protein and the correct, O-acetylated glycan structure. Basedon these results, it could also be predicted that the CP8-EPAbioconjugate consisted of the EPA protein and the correct, O-acetylatedglycan structure.

Example 6: S. aureus Protein Glycosylation and Product Characterization

To prove the versatility of the “in vivo” glycosylation to generateglycoconjugate vaccine candidates several carrier proteins were used assubstrate to be glycosylated with CP5. To further increase the immuneresponse of the bioconjugate vaccine against S. aureus, the carrierprotein EPA is exchanged by AcrA form C. jejuni and two proteins from S.aureus: Hla and ClfA. To be used as carrier proteins Hla and ClfA weremodify by the insertion of the bacterial N-glycosylation sites. Theprocess was performed as described in WO 2006/119987 generating fourversions for Hla H35L: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 16 and three for ClfA: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12.

For glycosylation of Hla H35L site 130 E. coli cells (W3110 ΔwaaLΔwecAwzzE ΔrmlB-wecG) comprising two expression plasmids: one for HlaH35L production (SEQ ID NO: 16), in which expression of the Hla H35Lcontaining the N-terminal signal peptide for periplasmic secretion, oneN-glycosylation site and a hexa HIS-tag for purification is undercontrol of the ParaBAD promoter, and secondly one for expression of theCP5 chimeric cluster and pglB (SEQ ID NO.: 17) were used. This systemcorresponds to the beforehand optimized 2 plasmids expression system ofCP5-EPA with an exchanged protein carrier expression plasmid. Cells weregrown in a 12-L bioreactor in rich medium to an OD_(600 nm)=30,expression of Hla was induced by the addition 0.2% arabinose. Cells werepelleted by centrifugation; the cells were washed and suspended in 0.2vol sucrose buffer, pelleted, and lysed by osmotic shock. Thespheroplasts were pelleted by centrifugation, and the periplasmicproteins in the supernatant were loaded on a Ni²⁺ affinitychromatography. Glycosylated (CP5-Hla) and unglycosylated Hla wereeluted from the affinity column by 0.5 M imidazole and loaded on ananionic exchange chromatography Proteins were eluted with a lineargradient from 0 to 0.7 M NaCl to separate CP5-Hla from Hla. Theresulting protein was separated by SDS PAGE and stained by Coomassie, ortransferred to nitrocellulose membranes and probed with either anti His,anti Hla, or anti CP5 antisera, as indicated (FIG. 14C). The results inFIG. 14C show the formation of glycoconjugate (CP5-Hla) by coomassie(left lane) and western blot using anti His (middle left lane) and antiHla (middle right) and anti CP5 (right) antisera.

The identity of Hla H35L with an engineered glycosylation site 130 wasconfirmed by in-gel trypsinization and MALDI-MS/MS.

To further show that the carrier protein is exchangeable forglycosylation by CP5 and CP8, C. jejuni AcrA protein was used as aglycosylation acceptor (see FIG. 14D). Using the 3 plasmid system (SEQID NO: 3, SEQ ID NO: 15, and SEQ ID NO: 27), the production strain forthis conjugate was W3110 ΔwaaL habouring the CP5 chimeric cluster (SEQID NO: 3), the PglB protein induced by IPTG (SEQ ID NO: 27) and the AcrA(SEQ ID NO: 15) under arabinose induction on separate plasmids. Cellswere grown in a bioreactor with a starting volume of 7 L in semi-definedmedium containing glycerol, peptone and yeast extract as C-sources.Cells were grown in batch or pulsed-batch mode to an OD_(600 nm) of 30,and expression of PglB and AcrA was induced by the addition of 1 mM IPTGand 10% arabinose. After induction, cells were further cultivated infed-batch mode for a period 15 hours under oxygen-limiting conditionsand collected by centrifugation. Cells were pelleted by centrifugation;the cells were washed and suspended in 0.2 vol sucrose buffer, pelleted,and lysed by osmotic shock. The spheroplasts were pelleted bycentrifugation, and the periplasmic proteins were loaded on a Ni²⁺affinity chromatography. CP5-AcrA glycoproteins were eluted from theaffinity column by 0.5 M imidazole. The purified protein was separatedby SDS PAGE and stained by Coomassie, or transferred to nitrocellulosemembranes and probed with either anti AcrA, or anti CP5 antisera, asindicated in FIG. 14D.

The insertion of the bacterial N-glycosylation sites in ClfA wasperformed as described in WO 2006/119987, generating SEQ ID NO: 10; SEQID NO: 11; SEQ ID NO: 12. The carrier proteins were expressed in E. colicells from arabinose inducible promoters. The genes were designed toproduce a N-terminal signal peptide for periplasmic secretion, severalN-glycosylation sites and a hexa HIS-tag for purification. Purificationwas started from periplasmic extracts of E. coli cells.

For glycosylation of ClfA 327 the beforehand optimized expressionsystems of CP5-EPA was employed. Using the 2 plasmid system (SEQ ID NO:17 and SEQ ID NO: 11), E. coli cells (W3110 ΔwecAwzzE ΔrmlB-wecG ΔwaaL)comprising the CP5 chimeric cluster and pglB (constitutive expressioncassette) as well as the expression plasmid for ClfA 327 (under controlof the ParaBAD promoter) were grown in 1 L Erlenmeyer flasks in LBmedium. An overnight culture was diluted to OD_(600 nm)=0.05. AtOD_(600 nm) around 0.5, ClfA expression was induced by addition ofarabinose (0.2% final concentration). The cells were grown for 20 hours.Cells were pelleted by centrifugation; the cells were washed andsuspended in 0.2 vol sucrose buffer, pelleted, and lysed by osmoticshock. The spheroplasts were pelleted by centrifugation, and theperiplasmic proteins were loaded on a Ni²⁺ affinity chromatography.ClfA-CP5 was eluted by 0.5M imidazole, was separated by SDS PAGE andstained by Coomassie, or transferred to nitrocellulose membranes andprobed with either anti His, or anti CP5 antisera. FIG. 14E shows theresults obtained using the ClfA variant with the glycosylation siteinserted around amino acid position 327 of the protein (SEQ ID NO: 11).They show the formation of ClfA by Coomassie staining and anti Hiswestern blot, and glycoconjugate (CP5-ClfA) by western blot using antiCP5 antisera.

Example 7: Activity of CP5-EPA as Glycoconjugate Vaccine

W3110 ΔwaaL ΔwecAwzzECA::cat cells comprising CP5 chimeric cluster (SEQID NO: 3) with cap5K inside, the PglB protein (SEQ ID NO: 27) and EPAwith signal 2 glycosylation sites on pEC415 (SEQ ID NO: 13) were grownin 1 L Erlenmeyer flasks in LB medium. An overnight culture was dilutedto OD_(600 nm)=0.05. At OD_(600 nm) around 0.5, EPA and PglB expressionwas induced by addition of arabinose (0.2% final concentration) and 1 mMIPTG, respectively. The cells were grown for 20 hours. Cells werepelleted by centrifugation; the cells were washed and suspended in 0.2vol sucrose buffer, pelleted, and lysed by osmotic shock. Thespheroplasts were pelleted by centrifugation, and the periplasmicproteins were loaded on a Ni²⁺ affinity chromatography. Glycosylated andunglycosylated EPA were eluted from the affinity column by 0.5 Mimidazole and loaded on a SourceQ anionic exchange column. GlycosylatedEPA was separated from unglycosylated EPA by applying a gradient ofincreasing concentration of NaCl. Eluted protein amounts were determinedby the BCA assay and based on the size of the bands obtained on SDS PAGEstained by Coomasie the theoretical mass of the sugar was calculated.Together with the protein determination, the amount of polysaccharideantigen was estimated in the preparation. This estimated quantificationwas confirmed by high mass maldi MS method (see FIG. 14A).

To measure the immunogenicity of CP5-EPA in living animals, 1 g of thepurified glycoconjugate was injected into mice by the IP (intraperitoneal) route in the presence of Aluminium hydroxide as adjuvant ondays 1 (first injection), 21 (second injection), and 56 (thirdinjection). After 35 and 61 days, which were two weeks after the secondand third injections, respectively, the IgG response was measured byELISA using a poly-L-lysine modified CP5 for coating (Gray, B. M. 1979.ELISA methodology for polysaccharide antigens: protein coupling ofpolysaccharides for adsorption to plastic tubes. J. Immunol.28:187-192). Blood from mice immunized with CP5-bioconjugate wasanalyzed for specific IgG antibodies against CP5 capsularpolysaccharide. FIG. 15A presents the IgG titers raised by CP5-EPA inmice. ELISA plates were coated with poly-L-lysine modified CP5, IgGresponse in mice immunized twice (second bar (empty) at each dilution)or three times (first bar (forward diagonals) at each dilution) withCP5-EPA was measured in triplicates. The signals obtained with thepreimmune sera as control are indicated by the third bar (backwarddiagonals) at each dilution. The mice IgG response was measured withalkaline phosphatase-conjugated protein G. As shown in FIG. 15A, theCP5-EPA bioconjugate elicited a serum antibody titer of 6.4×10³. Theresults presented in FIG. 15A show that CP5-EPA raises CP5 specificantibodies in mice. This experiment shows that the bioconjugate producedin E. coli is immunogenic in mice.

A similar experiment was performed in rabbits as the host organism.CP5-EPA (15 g CP5) was injected into rabbits intra-dermal in thepresence of Freund's complete adjuvant on day 1 and subcutaneously inthe presence of Freund's incomplete adjuvant on days 20, 30 and 40.After 61 days, the IgG response was measured by ELISA using apoly-L-lysine modified CP5 for coating (Gray, B. M. 1979. ELISAmethodology for polysaccharide antigens: protein coupling ofpolysaccharides for adsorption to plastic tubes. J. Immunol.28:187-192). FIG. 15B presents IgG titers raised by CP5-EPA in rabbits.The results presented in FIG. 15B show that CP5-EPA raises CP5 specificantibodies in rabbits. Immune response to CP5-EPA bioconjugate is thesecond bar (forward diagonals) at each dilution. Control sera includeCP5-specific absorbed sera raised to killed S. aureus (WC extracts,first bar (dots) at each dilution) and preimmune serum (third bar(empty) at each dilution). Serum from rabbits immunized with variousantigens was analyzed for specific antibodies to purified CP5. Plateswere coated with poly-L-lysine modified CP5. The signals obtained withthe preimmune sera as control are indicated by the third bar (backwarddiagonals) at each dilution. The rabbit IgG response was measured withalkaline phosphatase-conjugated protein G in triplicates. The CP5-EPAbioconjugate elicited a titer of 1×10⁶, which was 4 times higher thanthe titer of control sera (prepared by immunization with whole killed S.aureus and then absorbed with Wood 46 and a trypsinized isogenicacapsular mutant, so that the antiserum was rendered CP5-specific). Thisexperiment shows that the bioconjugate was able to elicit a high-titeredCP5-specific IgG response.

Example 8: Functional Activities of CP5 Antibodies

In Vitro Activity

The rabbit polyclonal antiserum raised as described in Example 7 waspurified by Protein A affinity column to enrich for IgG specificantibodies. IgG from rabbits immunized with S. aureus bioconjugateCP5-EPA was tested for functional activity in a classic in vitroopsonophagocytic killing assay (Thakker, M., J.-S. Park, V. Carey, andJ. C. Lee. 1998. Staphylococcus aureus serotype 5 capsularpolysaccharide is antiphagocytic and enhances bacterial virulence in amurine bacteremia model. Infect Immun 66:5183-5189). S. aureus wascultivated for 24 h on Columbia agar+2% NaCl. The bacteria weresuspended in minimal essential medium+1% BSA (MEM-BSA). PMNs(polymorphonuclear neutrophils) were isolated from fresh human blood,washed, counted, and suspended in MEM-BSA. The purified IgG preparationsfrom rabbits immunized with either S. aureus CP5-EPA or as controlpurified IgG preparations from rabbits immunized with Shigella O1-EPAthat has been purified as described in WO 2009/104074 were added to theassay in serial 10-fold dilutions prepared in MEM-BSA. Guinea pig serum(Pel-Freez) was used as a C′ source. Each assay (0.5 ml total volume)contained ˜5×10⁶ PMNs, 1×10⁶ CFU S. aureus, 0.5% to 1% guinea pig serum,and varying concentrations of IgG, ranging from 140 μg/ml to 1 μg/ml.Control samples contained 1) S. aureus incubated with C′ and PMNs, butno antibody; 2) S. aureus incubated with IgG and C′, but no PMNs; and 3)S. aureus alone. The samples were rotated end-over-end (12 rpm) for 2 hat 37° C. Sample dilutions were vortexed in sterile water, and bacterialkilling was estimated by plating the diluted samples in duplicate onTSA. The percent killing was defined as the reduction in CFU/ml after 2h compared with that at time zero.

In the first set of experiments, the opsonophagocytic killing of themethicillin-sensitive S. aureus (MSSA) strain Reynolds, the prototypeCP5 isolate, was tested, and the results are shown in FIG. 16A. Opsonicactivity of antibodies to CP5-EPA raised in rabbit was tested againstthe S. aureus serotype 5 strain Reynolds. CP5-EPA antibodies wereopsonic down to a concentration of 1.4 μg/ml, whereas O1-EPA antibodiesshowed little opsonic activity at 140 g/ml. A positive control serumraised against S. aureus whole cell extracts (obtained from J. C. Lee atthe Department of Medicine, Brigham and Women's Hospital, HarvardMedical School, Boston, Mass., USA) showed similar activity as the antiCP5-EPA serum (WC antiserum 1%).

As shown in FIG. 16A, between 65-75% of S. aureus Reynolds was killed byPMNs when incubated with antibodies to CP5-EPA and 1% guinea pig serumwith complement activity. The antiserum was used at a final 1% in theassay, and 89% of the S. aureus inoculum was killed under theseconditions. Little killing was observed when S. aureus was opsonized byC′ alone (1% guinea pig serum) or antibodies and C′ with no PMNs. Thedata shown are the means of 2 to 5 experiments. All samples graphedincluded guinea pig serum C′, and no killing was observed in the absenceof C′. Neither antibodies alone nor complement alone were opsonic, andthis feature is characteristic of encapsulated bacterial pathogens. Incontrast, antibodies elicited by the control vaccine (Shigella 01antigen coupled to EPA) did not show opsonic activity, even in thepresence of C′. As a positive control in this assay, we also testedCP5-specific rabbit antiserum (obtained from J. C. Lee at the Departmentof Medicine, Brigham and Women's Hospital, Harvard Medical School,Boston, Mass., USA). These data show that antibodies raised to theCP5-EPA bioconjugate showed opsonic activity against encapsulated S.aureus that is comparable to CP5 antibodies with documented opsonicactivity (Thakker, M., J.-S. Park, V. Carey, and J. C. Lee. 1998.Staphylococcus aureus serotype 5 capsular polysaccharide isantiphagocytic and enhances bacterial virulence in a murine bacteremiamodel. Infect Immun 66:5183-5189).

The opsonic activity of antibodies to CP5-EPA tested against the MRSAstrain USA100 of CP5-EPA. FIG. 16B presents the results of the opsonicactivity of IgG and C′ tested against S. aureus strain USA 100, a CP5+isolate, and is called NRS382. The data shown are the means of 2 to 5experiments. All samples graphed included guinea pig serum C′, and nokilling was observed in the absence of C′. As shown in FIG. 16B, ˜60% ofthe USA100 inoculum was killed by PMNs incubated with 0.5% guinea pigcomplement and concentrations of CP5-EPA IgG ranging from 100 to 1μg/ml. Minimal killing was observed in the absence of PMNs or when IgGwas omitted from the assay. No killing was achieved when IgG raised tothe O1-EPA conjugate vaccine was added to PMNs+C′ (the bacteriamultiplied in this sample). Little killing was observed when S. aureuswas opsonized by C′ alone or antibodies and C′ with no PMNs. Thus,CP5-EPA antibodies were opsonic at concentrations ranging from 100 to 1μg/ml, whereas O1-EPA antibodies showed little opsonic activity at 100μg/ml. This experiment shows that CP5-EPA antibodies display opsonicactivity against both MSSA and MRSA strains.

In Vivo Activity

To determine whether the opsonic activity of IgG raised to thebioconjugate CP5-EPA vaccine would predict protection in a mouse modelof staphylococcal infection, passive immunization experiments wereperformed. In the initial studies, Swiss-Webster male mice (˜6 wks ofage) were injected IV (tail vein) with 1.4 to 2 mg IgG from rabbitsimmunized with CP5-EPA or Shigella O1-EPA. After 24 h, the mice werechallenged by the intra-peritoneal (IP) route with ˜3.6×10⁷ CFU S.aureus Reynolds. Bacteremia levels were measured 2 h after challenge toassess antibody-mediated clearance of the bacteremia. The lower limit ofdetection by culture was 5 CFU/ml blood. FIG. 17A show the resultingbacteremia levels. Each dot represents a quantitative blood cultureperformed by tail vein puncture on an individual mouse 2 h afterbacterial inoculation. Horizontal lines represent median CFU/ml values.Empty circles are blood samples from mice that obtained anti CP5-EPAantibodies, black filled circles are samples from animals that got acontrol antibody preparation which was raised against EPA conjugated toa different glycan (S. dysenteriae O1). The results of FIG. 17A showthat mice given CP5 antibodies showed a significant (P=0.0006 byMann-Whitney analysis) reduction in bacteremia levels compared to micegiven the O1-specific antibodies. In fact, the reduction in CFU/ml bloodwas 98% in mice passively immunized with the CP5-EPA vs. mice givenO1-EPA IgG.

In subsequent passive immunization experiments, mice were challenged IPwith a lower inoculum (˜5.5×10⁶ CFU/mouse) of S. aureus Reynolds.Passive immunization with CP5-EPA antibodies was tested in micechallenged IP with 5-6×10⁶ CFU S. aureus Reynolds. Mice were injectedintravenously (IV) with 2 mg CP5-EPA IgG or 01-EPA IgG 24 h beforebacterial challenge. FIG. 17B shows the resulting bacteremia levels.Each dot represents a quantitative blood culture performed by tail veinpuncture on an individual mouse 2 h after bacterial inoculation.Horizontal lines represent median CFU/ml values. Empty circles are bloodsamples from mice that obtained anti CP5-EPA antibodies, black filledcircles are samples are from animal that got a control antibodypreparation which was raised against EPA conjugated to a differentglycan (S. dysenteriae 01). As shown in FIG. 17B, mice given 2 mgCP5-EPA IgG had significantly (P<0.0001 by Mann-Whitney analysis) lowerbacteremia levels than animals given 2 mg of O1-EPA IgG. In fact, 6 of 7mice passively immunized with CP5-EPA antibodies had sterile bloodcultures (lower limit of detection 6 to 30 CFU/ml blood, depending onthe blood volume collected and plated from each mouse). The reduction inbacteremia levels attributable to CP5 antibodies was 98%, compared tocontrol mice given O1-EPA IgG.

To determine whether protection against bacteremia could be conferred bya lower level of IgG, a subsequent experiment was performed wherein micewere passively immunized by the IV route with 300 g CP5-EPA or 01-EPAIgG. After 24 h, the mice were inoculated IP with 6×10⁶ CFU S. aureusReynolds. The lower limit of detection by culture was 13-67 CFU/mlblood. FIG. 17B shows the resulting bacteremia levels. Each dotrepresents a quantitative blood culture performed by tail vein punctureon an individual mouse 2 h after bacterial inoculation. Horizontal linesrepresent median CFU/ml values. Empty circles are blood samples frommice that obtained anti CP5-EPA antibodies, black filled circles aresamples are from animal that got a control antibody preparation whichwas raised against EPA conjugated to a different glycan (S. dysenteriae01). As in FIG. 17B, the results of FIG. 17C show CP5 antibody-mediatedprotection against bacteremia was achieved with this lower antibodydose. A 98% reduction in bacteremia levels was achieved by antibodieselicited by the CP5 bioconjugate vaccine, and 8 of 9 mice had sterileblood cultures compared to 0 of 8 mice given Shigella 01-EPA antibodies.

Example 9: Active Immunization in Mice

To show that vaccination of mice with the bioconjugate CP5-EPA mediatesprotection against bacterial challenge as in passive immunization assay,active immunization studies were performed.

CP5-EPA bioconjugate was produced in E. coli W3110 ΔwaaL ΔwecAwzzE::catby co-expression of the chimeric CP5 gene cluster (SEQ ID NO: 3), PglB(SEQ ID NO: 27) from plasmid pEXT21 and EPA (containing twoglycosylation sites, SEQ ID NO: 13) from separate plasmids. Cells weregrown in a bioreactor with a starting volume of 7 L in semi-definedmedium containing glycerol, peptone and yeast extract as C-sources.Cells were grown in batch or pulsed-batch mode to an OD_(600 nm) of 30,and expression of PglB and EPA was induced by the addition of 1 mM IPTGand 10% arabinose. After induction, cells were further cultivated infed-batch mode for a period 15 hours under oxygen-limiting conditionsand collected by centrifugation. The cells were washed and resuspendedin 25% sucrose buffer at an OD_(600 nm)=200, pelleted, and lysed byosmotic shock. The spheroplasts were pelleted by centrifugation, and theperiplasmic proteins were loaded on a Ni²⁺ affinity chromatography.Glycosylated and unglycosylated EPA were eluted from the affinity columnby 0.5 M imidazole and loaded on a SourceQ anionic exchange column.Glycosylated EPA was separated from unglycosylated EPA by applying agradient of increasing concentration of NaCl.

CP5-EPA is intended to be used as a conjugate vaccine to protect againstCP5 S. aureus strains. To test whether such active immunization isfunctional, we immunized different groups of female Swiss Webster micewith three different doses of CP5-EPA and analyzed the immunizationusing a bacteremia model. Three doses were subcutaneously injected atdays 0, 14 and 28. Mice were intra-peritoneally challenged at day 42with S. aureus strain JL278, as shown in FIG. 18. Five groups of micewere immunized with three different doses of CP5-EPA as indicated belowthe x-axis (dotted circles; empty circles; and backward diagonals incircles). Two control groups received either adjuvants (forwarddiagonals in circles) or PBS (black filled circles) alone. Each dotrepresents a blood sample from a single mouse. The lowest dose ofvaccine (0.2 μg) induced protection in all mice from the group. Twohours after challenge blood samples were analyzed for cfu formation andanti CP5 antibodies by ELISA using a poly-L-lysine modified CP5 forcoating (Gray et al. (1979)). In all groups immunized with CP5-EPA, amean reduction of cfu in blood was observed. However, only in the groupwhich received the lowest dose of vaccine, there was a generalprotection from bacteremia in all five mice. Analysis of blood for antiCP5 antibodies resulted in a positive correlation of protection and meanELISA titers in the different mouse groups. The results presented inFIG. 18 indicate that the antibodies induced the protection frombacteremia in immunized mice.

These studies indicate that the CP5-EPA bioconjugate vaccine inducedantibodies that opsonized S. aureus for phagocytic killing by human PMNsand protected mice against bacteremia in positive and activeimmunization studies. These data provide strong evidence that thepresented bioconjugate will protect against disease provoked by multipleS. aureus strains.

While this invention has been particularly shown and described withreferences to embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and detail may be madetherein without departing from the scope of the invention encompassed bythe claims.

1. A Gram-negative host prokaryotic organism comprising: (i) anucleotide sequence encoding at least one glycosyltransferase from aGram-positive bacterium; (ii) a nucleotide sequence encoding at leastone glycosyltransferase from a Gram-negative bacterium; (iii) anucleotide sequence encoding a carrier protein; and (iv) a nucleotidesequence encoding an oligosaccharyl transferase.
 2. The host prokaryoticorganism of claim 1, wherein the Gram-negative bacterium produces apolysaccharide that has structural similarity to a second polysaccharideproduced by the Gram-positive organism.
 3. The host prokaryotic organismof claim 2, wherein the polysaccharide produced by the Gram-negativebacterium contains a repeating unit that is partially identical to arepeating unit produced by the Gram-positive organism.
 4. The hostprokaryotic organism of claim 1, wherein the protein comprises at leastone inserted consensus sequence D/E-X-N-Z-S/T, wherein X and Z may beany natural amino acid except proline.
 5. The host prokaryotic organismof claim 1, wherein the protein comprises at least two insertedconsensus sequence D/E-X-N-Z-S/T, wherein X and Z may be any naturalamino acid except proline.
 6. The host prokaryotic organism of claim 1,wherein said host organism is selected from the group consisting ofEscherichia ssp., Campylobacter ssp., Salmonella ssp., Shigella ssp.,Helicobacter ssp., Pseudomonas ssp. or Bacillus ssp.
 7. The hostprokaryotic organism of claim 1, wherein said host cell organism isselected from the group consisting of Escherichia coli, Campylobacterjejuni, and Salmonella typhimurium.
 8. The host prokaryotic organism ofclaim 7, wherein said host organism is E. coli.
 9. A method of producinga glycosylated protein in the host prokaryotic organism of claim
 1. 10.The method of claim 9 wherein the glycosylated protein is a proteinN-glycosylated with capsular polysaccharide of a Gram positivebacterium.
 11. The method of claim 10 wherein the protein N-glycosyatedwith capsular polysaccharide of a Gram positive bacterium is synthesizedby a combination of different glycosyltransferases from differentorganisms, for example a Gram positive bacterium and a Gram positivebacterium.
 12. A method of producing a bioconjugate vaccine by producingin Gram-negative bacteria modified capsular polysaccharides onundecaprenol (Und), and linking these polysaccharide antigens to aprotein carrier comprising at least one inserted consensus sequenceD/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid exceptproline.
 13. A method of modifying a bacterium of a first Gram-negativespecies comprising: (i) selecting a Gram-positive bacterium as a target;(ii) identifying a first repeating unit of a polysaccharide produced bysaid Gram-positive bacterium comprising at least three monomers; (iii)identifying a polysaccharide produced by a bacterium of a secondGram-negative species comprising a second repeating unit comprising atleast two of the same monomers as said first repeating unit; (iv)inserting into said bacterium of a first Gram-negative species one ormore nucleotide sequences encoding glycosyltransferases that assemble atrisaccharide containing: a.) said second repeating unit; and b.) amonomer of said first repeating unit not present in said secondrepeating unit; (v) inserting a nucleotide sequence encoding a carrierprotein; and (vi) inserting a nucleotide sequence encoding anoligosaccharyl transferase.
 14. The method of claim 13, wherein saidprotein comprises at least one inserted consensus sequenceD/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid exceptproline.
 15. The method of claim 14, wherein said Gram-positivebacterium is S. aureus.